Optical detection of macromolecular heparin via selective coextraction into thin polymeric films

Thin plasticized polymer films, poly(vinyl chloride) doped with a specific ion pairing quaternary ammonium compound, tridodecylmethylammonium chloride, and a lipophilic pH indicator, 3-hydroxy-4-(4-nitrophenylazo)phenyl octadeconate, are shown to exhibit significant and analytically useful optical response toward macromolecular heparin. The response mechanism is based on favorable extraction of heparin into the bulk organic film, owing to the specific ion-pairing complexation reaction between the quaternary ammonium species and the polyanion. A simultaneous coextraction of hydrogen ions results in protonation of the pH chromophore and hence a change in the optical absorbance of the polymeric film. When used in a limited volume/fixed exposure (10 min) detection mode, film absorbances change as a function of the initial heparin concentration in the range of 0.2-3.0 units/mL (1.2-18 micrograms/mL). The practical measurement response time is controlled by heparin diffusion through the stagnant diffusion layer adjacent to the surface of the film as well as within the bulk of the polymer film and is shown to increase with the molecular weight of the heparin species tested. No optical response to heparin is observed when a strong heparin complexing agent (e.g., protamine) is present in the test solution, suggesting that the polymer film can be used to conveniently monitor heparin-protamine (or other antagonist) titrations. The theory relating to the operation of the sensing film in either the equilibrium or the kinetic mode and the selectivity of the optimized film to heparin relative to small anions are presented.     

Reduction of renal uptake of monoclonal antibody fragments by amino acid infusion

The renal uptake of radiolabeled antibody fragments and peptides presents a problem in radioimmunodetection and therapy, compromising lesion sensitivity, especially with intracellularly-retained isotopes. Previously, we showed that cationic amino acids and their derivatives are capable of significantly reducing kidney uptake in animals. We report our initial clinical results of successful renal uptake reduction in five patients who underwent cancer radioimmunodetection with 99mTc-anti-CEA Fab' fragments. METHODS: The patients were infused with two liters of a commercially-available nutritive amino acid solution (containing approximately 2.25 g/liter lysine-glutamate and 2.50 g/liter arginine), whereas 75 control patients received the same volume of saline (quantification of organ and tumor kinetics from conjugate whole-body views by ROI technique). RESULTS: The renal uptake in the amino acid group was significantly lower (p<0.05) than in the control group (11.1 +/- 2.0% injected dose versus 17.7 +/- 7.0% injected dose at 24 hr postinjection), whereas the uptake of all other organs remained unaffected. Gel filtration chromatography of the urine taken from amino-acid-treated patients showed that a significantly higher amount of excreted activity was bound to intact Fab' (53% of excreted activity) in contrast to only less than 10% in the control group. CONCLUSION: The renal uptake of monoclonal antibody fragments in patients can be reduced significantly by amino acid infusion, even at considerably lower doses than those that were safe and effective in animals. As was found in animals, the mechanism seems to rely on an inhibition of the re-absorption of tubularly-filtered proteins by the proximal tubule cells. These results encourage further clinical trials to lower the renal uptake experienced in radioimmunodetection, as well as in therapeutic trials with antibody fragments and peptides.     

Echinococcus granulosus: cloning of a thioredoxin peroxidase

Disseminated histoplasmosis has been recognized as a serious opportunistic infection in patients with acquired immunodeficiency syndrome (AIDS). However, cases reported in the literature have been predominantly in adult patients. Here we report an infant with AIDS who presented with fever, cough, rhinorrhea, hepatosplenomegaly, pancytopenia and coagulopathy, and died of respiratory failure. Autopsy revealed disseminated histoplasmosis involving multiple organs including lungs, intestines, liver, spleen, bone marrow, lymph nodes, kidneys, and meninges. The diagnosis was established based on histomorphology and confirmed by blood culture.     

Completed suicide among adolescents with no diagnosable psychiatric disorder

The characteristics of male adolescent suicide victims with (n = 84) and without (n = 8) a diagnosable psychiatric disorder were compared. Using psychological autopsy methods--interviews with victims' family members and health care professionals, and review of records (e.g., medical, school, police)-data were collected on all adolescent suicides in Finland during a 12-month period. Compared with adolescents with a psychiatric disorder, those with no disorder tended to come from less disturbed families, had shown less antisocial behavior, and had less frequently utilized health care and social services. Adolescents with no disorder more often communicated suicidal thoughts for the first time just before the suicide, and difficulties with the law (discipline problems) were more common precipitants than among those with a disorder. It was concluded that the process leading to suicide seems to be relatively short among male adolescents with no diagnosable psychiatric disorder. Communication of suicidal intent and problems with discipline are among the few clinical warning signs.     

Transient central hypothyroidism as a cause of failure to thrive in newborns and infants

The course of two neonates and one 4-month-old infant with laboratory and clinical evidence of central hypothyroidism is described. All three presented with failure to thrive and improved after L-T4 therapy. Early recognition and treatment of newborns and infants with central hypothyroidism is important to maximize the potential for growth and development. Two of the three infants have been documented to have transient central hypothyroidism of hypothalamic origin, not previously reported.     

Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability

The possibility of primitive hematopoietic cell ex vivo expansion is of interest for both gene therapy and transplantation applications. The engraftment of autologous rhesus peripheral blood (PB) progenitors expanded 10 to 14 days were tracked in vivo using genetic marking. Stem cell factor (SCF)/granulocyte colony-stimulating factor (G-CSF)-mobilized and CD34-enriched PB cells were divided into two equal aliquots and transduced with one of two retroviral vectors carrying the neomycin-resistance gene (neo) for 4 days in the presence of interleukin-3 (IL-3), IL-6, and SCF in the first 5 animals, IL-3/IL-6/SCF/Flt-3 ligand (FLT) in 2 subsequent animals, or IL-3/IL-6/SCF/FLT plus an autologous stromal monolayer (STR) in the final 2. At the end of transduction period, one aliquot (nonexpanded) from each animal was frozen, whereas the other was expanded under the same conditions but without vector for a total of 14 days before freezing. After total body irradiation, both the nonexpanded and expanded transduced cells were reinfused. Despite 5- to 13-fold higher cell and colony-forming unit (CFU) doses from the expanded fraction of marked cells, there was greater short- and long-term marking from the nonexpanded cells in all animals. In animals receiving cells transduced and expanded in the presence of IL-3/IL-6/SCF/FLT, engraftment by the marked expanded cells was further diminished. This discrepancy was even more pronounced in the animals who received cells transduced and expanded in the presence of FLT and autologous stroma, with no marking detectable from the expanded cells. Despite lack of evidence for expansion of engrafting cells, we found that the addition of FLT and especially STR during the initial brief transduction period increased engraftment with marked cells into a clinically relevant range. Levels of marked progeny cells originating from the nonexpanded aliqouts were significantly higher than that seen in previous 4 animals receiving cells transduced in the presence of IL-3/IL-6/SCF, with levels of 10% to 20% confirmed by Southern blotting from the nonexpanded IL-3/IL-6/SCF/FLT/STR graft compared with 0.01% in the original IL-3/IL-6/SCF cohort. These results suggest that, although expansion of PB progenitors is feasible ex vivo, their contribution towards both short- and long-term engraftment is markedly impaired. However, a brief transduction in the presence of specific cytokines and stromal support allows engraftment with an encouraging number of retrovirally modified cells.