Elimination of I131-labelled heterologous antigen from the blood of chickens after total body X-irradiation

A study was made on the effect of ionizing radiation upon the rate of elimination of I¹³¹. labelled human serum albumin (HSA I¹³¹) from the blood and upon antibody formation in chickens irradited with 1,2 00R(i.e.with LD₅₀) and injected with antigen 30 min, 6 days or 14 days after irradiation. The elimination curve from unirradiated control birds followed the typical three-phase pattern. The effect of irradiation was most marked with chickens injected with antigen 6 days after irradiation, resulting in an extension of the second phase with practically no third phase at all. Exposure to irradiation 30 min prior to antigen administration resulted in an extension of the second phase by 2 days as compared to the controls, with the onset of the third phase occurring on day 7. Irradiation 14 days prior to antigen administration resulted in an extension of the second phase by 1 day as compared to the controls, with the onset of the third phase occurring on day 6. Elimination of HSA I¹³¹ in the second phase was more rapid than that of I¹³¹-labelled chicken serum albumin (CSA I¹³¹) no matter whether the chickens were irradiated or not. This suggests that the capacity of specific antigen uptake is not affected by irradiation. Antigen elimination curves from control irradiated groups given CSA I¹³¹ followed the same pattern as that found in control unirradiated birds injected with homologous antigen.     

Elimination of Homologous And Heterologous (Bovine) Serum Albumin from the Blood Circulation in the Dog)

The elimination of intravenously administered I131-labelled bovine serum albumin has been compared to the elimination rate of relabelled homologous serum albumin in normal and bled dogs, which had lost considerable blood volumes. The investigation shows that during the first four to five days after the administration the elimination is similar of heterologous and homologous serum albumin. This proves that bovine serum albumin can be regarded to be an equivalent plasma expander to homologous serum albumin in the dog. Elimination of homologous as well as heterologous serum albumin follows a simple exponential curve during four to five days after administration. The intravascular half-lives for homologous serum albumin were 6.4 ±1.5 days and 6.4 ± 0.6 days respectively in control and bled dogs. Corresponding values for heterologous (bovine) serum albumin were 5.0 ± 0.3 and 4.8 ± 0.4 days respectively. The quote for cencentrations of homologous and heterologous serum albumin in different tissues was found to be relatively constant approximately 1.4. An exception was the stomach wall in bled dogs which had a quote of 1.1 only.     

The Lack of Selective Reincorporation into Tissue Protein of the Amino Acids Derived from the Catabolism of Serum Protein

Homologous S³⁵-labeled albumin, gamma globulin, and alpha-beta globulin were transfused into rabbits and the specific activities of the electrophoretic fractions of the sera of the recipients were determined at various time intervals up to 12 days after injection. Detectable reincorporation into a fraction other than that transfused was found only in the gamma globulin fraction after albumin injection. This activity rose between 2 and 12 days and reached a level of 2 to 3 per cent of the extrapolated zero time activity of the albumin fraction. When homologous serum protein doubly labeled with I¹³¹ and S³⁵ was transfused into mice, marked drops in the ratios of I¹³¹ to S³⁵ in the serum and tissue proteins were observed between 1 and 48 hours after injection. On the basis of a determination of the absolute and relative amounts of I¹³¹ and S³⁵ found in the various tissue and serum proteins, the amount of reincorporation of S³⁵ into each protein was calculated. The relative amounts of reincorporation of S³⁵ among the various tissues were remarkably similar to the relative amounts of incorporation of S³⁵ after the injection of labeled free amino acids. It is concluded that serum protein does not form a major direct source of amino acids to the tissues but feeds them indirectly through the extracellular pool.     

The Purity of Radioiodide-I131 Assessed by in Vivo and in Vitro Methods

Between 41 and 94% of the radioactivity of 24 preparations of I¹³¹ supplied without cysteine preservative was non-iodide on chromatographic analysis. Extraneous radio-activity was essentially absent from I¹³¹ supplied with cysteine. It was converted to iodide-I¹³¹ by 10^-3 M cysteine or iodide but not by incubation at pH 2. The average thyroid uptake of I¹³¹ containing extraneous radioactivity was significantly lower than the uptake of I¹³¹ free from non-iodide impurity in 16 human subjects measured under controlled conditions and in a random group of 669 patients. Incubation of samples of I¹³¹ containing non-iodide radioactivity with tyrosine and cupric chloride resulted in the non-enzymatic formation of monoiodotyrosine-I¹³¹ either in the presence or absence of thyroid homogenate. Enzymatic formation of monoiodotyrosine-I¹³¹ by thyroid homogenates could be demonstrated only when I¹³¹ free from extraneous activity was used.     

Fibroblast-like cells from embryonic chick cornea, heart, and skin are antigenically distinct.

Populations of fibroblast-like cells from 14 day embryonic chick cornea, heart, and skin were grown in vitro as primary cultures and found to be antigenically distinct from one another. Corneal fibroblasts were obtained by dissection, whereas heart and skin fibroblast-like cells were separated from nonfibroblastic cell types by their rapid adhesion to substrata. Cultured cells were used as antigens in rabbits. Antisera were first absorbed against homogenates of embryonic chicks from which the homologous tissue was removed. Each such 1° absorbed antiserum then was absorbed against homogenates of the two respective heterologous fibroblast-like cell populations (2° and 3° absorptions). Resulting 3° absorbed antisera were tested for specificity by immunodiffusion, immune agglutination, immune cytotoxicity (trypan blue uptake and ⁵¹Cr release), and indirect immunofluorescence. Each 3° antiserum was judged tissue specific when it reacted only with the fibroblast-like cells of its own tissue, i.e., the homologous population. Unabsorbed antisera reacted with both homologous and heterologous fibroblast-like cells, as did 1° absorbed antisera. Absorption of 1° antisera with homogenates of the two heterologous fibroblast-like populations removed antibodies against the heterologous populations without significantly reducing the 3° antiserum titer against the homologous fibroblast cell type. Moreover, absorption of 1° antisera with each of the two heterologous fibroblast-like populations removed antibodies not removed by the other. Thus, the fibroblast-like cells from cornea, heart, and skin are antigenically different from one another in vitro. The stable antigenic differences detected may have arisen during the differentiation of these cells in vivo. Some of the tissue-specific antigens detected must occur on the cell surface.     

Trypanosoma cruzi: immunosuppressed response to different antigens in the infected mouse.

Trypanosoma cruzi: Immunosuppressed response to different antigens in the infected mouse. Experimental Parasitology45, 190–199. Trypanosoma cruzi infection in mice results in functional changes in the normal immunological responses to heterologous antigens. An immunosuppression of the 19 and 7S antibody response is observed in infected animals against both a particulate antigen and against soluble antigens. Furthermore, the immune response to the soluble T-independent antigens, DNP-Ficoll and LPS, was also similarly impaired when antigen was administered to trypanosome-infected animals. The suppression of the immune response to these antigens does not seem to involve an alteration in the macrophage, as evidenced by a normal uptake and handling of soluble ¹³¹I-labeled HSA and by a normal immune response when antigen-exposed peritoneal macrophages from trypanosome-infected mice were transferred to normal mice. These data support the concept that T. cruzi induces an immunosuppression to both T-dependent and T-independent antigens and that the depression observed is not due to an alteration in macrophage function.     

Calibration line determination of an heterologous antigen in radial immunodiffusion

Using known principles of radial immunodiffusion it is shown experimentally that an heterologous antigen may be quantified using an homologous antigen-antibody system. The proteins in which this is demonstrated are human serum albumin (the homologous antigen) and the plasma albumins of baboon (Papio hamadryas) and rhesus monkey (Macaca mulatta). Purification of the heterologous antigen is not required. The system also gives a measure of the degree of cross-reactivity between the homologous and heterologous antigens.     

Separation of antigens by immunological specificity: Use of disulphide-linked antibodies as immunosorbents

1. γ-Globulin concentrates of antisera prepared against ovalbumin and human serum albumin were thiolated and cross-linked to form insoluble polymers. 2. These immunosorbents were of low solubility and of high capacity for homologous antigen. 3. The high specificity of these immunosorbents was demonstrated by fractionation of various binary mixtures of fluorescent ovalbumin, ¹³¹I-labelled human serum albumin, lysozyme and ribonuclease.     

MATURATION OF THE RAT FETAL THYROID

Maturation of the rat fetal thyroid was studied with the aid of I¹³¹ and of fluorescence and electron microscopy. The I¹³¹ concentration of the fetal gland increased exponentially from day 17 to day 20 of gestation and was related to the weight of the fetus (and presumably the weight of the thyroid) and also to the quantity of I¹³¹ accumulated by the fetus. In the 17-day gland, thyroglobulin or immunologically similar material was sparsely present in the incipient lumens of some cell clusters. With maturation, this material increased and was also observed within follicular cells on days 18 to 19 of gestation. On day 20, the specifically reacting material was present in the follicular lumens and was absent from the cytoplasm of follicular epithelium. Ultrastructurally, the earliest thyroid cells examined were replete with all the organelles found in the more mature epithelium. No direct correlation could be made between the cytoplasmic structures and the presence of thyroglobulin, although the granular endoplasmic reticulum was most likely the organelle responsible for synthesis of thyroglobulin. Thyroglobulin or a precursor was found in fetal thyroid cells before measurable quantities of I¹³¹ were concentrated and before cytoplasmic droplets appeared.     

The Role of the Gut in Albumin Catabolism : I. Studies in the jejunoileectomized rabbit

I¹³¹-albumin metabolic studies were carried out in 5 rabbits before, 3 weeks after, and several months after removal of 70 to 90 per cent of the jejunum and ileum. A sixth animal was studied before and 11 weeks after a sham operation. During postoperative experiments, the animals were found to be in a highly unsteady state with large losses of albumin from the vascular compartment. Despite these losses, the plasma albumin concentration was maintained at a relatively constant level. No decrease in the albumin efflux occurred following nearly complete jejuno-ileectomy. The data suggest that albumin catabolism is a first order process.     

HYPERTHYROIDISM—Diagnosis by Failure of Triiodothyronine to Suppress Uptake of Iodine131

Sixty patients with abnormally high I¹³¹ uptake were treated with liothyronine (L-triiodothyronine) for seven or eight days and then tested again. Fifty-five patients showed a suppression of iodine uptake sufficient to eliminate the possibility of hyperthyroidism. Also the therapeutic trial of liothyronine indicated whether they were euthyroid, or hypothyroid with iodine deficiency. Six of the patients showed insignificant change in the I¹³¹ uptake after treatment with thyroid hormone—that is, the change from the original uptake was less than 30 per cent. These six patients were later confirmed to be hyperthyroid. In four patients the uptake at the second test was less by between 35 and 52 per cent than at the first. These four patients, like the remainder with even greater suppression in thyroid uptake, remained clinically euthyroid.The repeat I¹³¹ uptake test was thus seen to be considerably more accurate than the single test and well worth the time required to perform it.     

Antibody formation in young rabbits immunized with heterologous serum proteins

Rabbits were immunized with human or bovine albumin at different intervals after birth and antibody formation was studied by haemagglutination of red cells sensitized with the relevant antigen. The intraperitoneal injection of antigen in amounts of 5 mg. induced antibody formation in some litters 16–20 days after immunization, if the animals were over three days old when immunized. In younger rabbits the same dose induced tolerance. Even when different methods of enhancing the effect of the antigen (Freund’s adjuvant, Al (OH)₃, antigen-conjugated red cells, immune precipitates) or very small doses of antigen were used, antibody formation was still not detected before the 20th day of life. The use of¹³¹I-BSA did not demonstrate the immune phase of elimination of the antigen during 17 days after administration of the antigen, even in rabbits immunized 14 days after birth. The relationship of antibody formation to the induction of tolerance and the difference in the response of newborn rabbits to immunization with the different types of antigen is discussed.     

Comparison of the biodistribution of 75Se- and 131I-labelled monoclonal antibodies in nude mice

A monoclonal antibody, C-215, against colon cancer, was internally labelled with [⁷⁵Se]methionine. The biodistribution was studied in tumour-bearing nude mice and compared with the biodistribution of [¹³¹I]C-215. The tissue uptake was divided into three parts: antibody bound to the antigen, antibody in the extracellular space and uptake of the released radionuclide. [⁷⁵Se]C-215 showed a greater amount of antigen-bound antibody in the tumour, but also a greater unspecific uptake both in tumour and normal tissue.     

The significance of extrathyroidal radioactive iodine accumulation and secretion in clinical pathology

Summary The uptake and the distribution of ¹³¹I^– injected intravenously into rats was followed up by whole body autoradiography. Extrathyroidal accumulations with some interest for clinical pathology were found in the nose, the hair follicles and the mucous cells of the stomach.     

A Comprehensive Insight into Binding of Hippuric Acid to Human Serum Albumin: A Study to Uncover Its Impaired Elimination through Hemodialysis

Binding of hippuric acid (HA), a uremic toxin, with human serum albumin (HSA) has been examined by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), molecular docking, circular dichroism (CD) and fluorescence spectroscopy to understand the reason that govern its impaired elimination through hemodialysis. ITC results shows that the HA binds with HSA at high (K _b ∼10⁴) and low affinity (K _b ∼10³) sites whereas spectroscopic results predict binding at a single site (K _b∼10³). The HA form complex with HSA that involves electrostatic, hydrogen and hydrophobic binding forces as illustrated by calculated thermodynamic parameters. Molecular docking and displacement studies collectively revealed that HA bound to both site I and site II; however, relatively strongly to the later. Esterase-like activity of HSA confirms the involvement of Arg410 and Tyr411 of Sudlow site II in binding of HA. CD results show slight conformational changes occurs in the protein upon ligation that may be responsible for the discrepancy in van’t Hoff and calorimetric enthalpy change. Furthermore, an increase in and is observed from DSC results that indicate increase in stability of HSA upon binding to HA. The combined results provide that HA binds to HSA and thus its elimination is hindered.     

Radioiodine treatment for graves’ disease: a 10-year Australian cohort study

Background

Radioactive iodine (I¹³¹) is a common definitive treatment for Graves’ Disease. Potential complications include worsening, or new development of Graves’ eye disease and development of a radiation thyroiditis. The purpose of the present study was to assess outcomes of patients treated with I¹³¹ in an Australian tertiary centre over 10 years.

Methods

Data from 101 consecutive patients treated with I¹³¹ for a diagnosis of Graves’ disease between 2005 to 2015 was collected and reviewed retrospectively. Baseline TSH receptor antibody titre, pre-treatment free thyroxine (FT4), technetium scan uptake, initial treatment, duration of treatment, reason for definitive therapy, complications, and time to remission (defined as euthyroidism or hypothyroidism after 12 months) were recorded.

Results

Of the 92 patients with adequate outcome data, 73 (79.3%) patients achieved remission with a single dose of I¹³¹. Of the remaining 19 patients, 12 had a second dose and became hypothyroid. TSH receptor antibody titre at diagnosis was significantly lower in the group that achieved remission with the first dose compared with those who did not (P =?0.0071). There was no difference in technetium uptake, I¹³¹ dose, duration of therapy or pre-treatment free thyroxine (FT4). I¹³¹ was complicated by development of eye disease in 3 patients and 1 (of 11 with pre-existing eye disease) had worsening eye disease. A clinically apparent flare of hyperthyroidism following I¹³¹ was evident in 8 patients (8.6%).

Conclusion

Radioiodine is an effective therapy for Graves’ Disease with few complications. The majority of patients achieve remission with a single dose. Those who require a second dose are more likely to have higher TSH receptor antibody titres at diagnosis. To the best of our knowledge, this is the first study to report outcomes from radioiodine treatment for Graves’ disease in an Australian population.

     

Insight into the interaction of antitubercular and anticancer compound clofazimine with human serum albumin: spectroscopy and molecular modelling

The binding of clofazimine to human serum albumin (HSA) was investigated by applying optical spectroscopy and molecular docking methods. Fluorescence quenching data revealed that clofazimine binds to protein with binding constant in the order of 10⁴ M^?1, and with the increase in temperature, Stern–Volmer quenching constants gradually decreased indicating quenching mode to be static. The UV–visible spectra showed increase in absorbance upon interaction of HSA with clofazimine which further reveals formation of the drug–albumin complex. Thermodynamic parameters obtained from fluorescence data indicate that the process is exothermic and spontaneous. Forster distance (R_o) obtained from fluorescence resonance energy transfer is found to be 2.05 nm. Clofazimine impelled rise in α-helical structure in HSA as observed from far-UV CD spectra while there are minor alterations in tertiary structure of the protein. Clofazimine interacts strongly with HSA inducing secondary structure in the protein and slight alterations in protein topology as suggested by dynamic light scattering results. Moreover, docking results indicate that clofazimine binds to hydrophobic pocket near to the drug site II in HSA.     

Endothelial cell leptin receptor mutant mice have hyperleptinemia and reduced tissue uptake

Hyperleptinemia is usually associated with obesity and leptin resistance. Endothelial cell leptin receptor knockout (ELKO) mice without a signaling membrane‐bound leptin receptor in endothelia, however, have profound hyperleptinemia without signs of leptin resistance. Leptin mRNA in adipose tissue was unchanged. To test the hypothesis that the ELKO mutation results in delayed degradation and slowed excretion, we determined the kinetics of leptin transfer in groups of ELKO and wildtype mice after intravenous bolus injection of ¹²⁵I‐leptin and the reference substance ¹³¹I‐albumin. The degradation pattern of ¹²⁵I‐leptin in serum and brain homogenates at different time points between 10 and 60 min was measured by HPLC and acid precipitation. Although ELKO mice had reduced uptake of ¹²⁵I‐leptin uptake by the brain and several peripheral organs, leptin was more stable in blood and tissue. There was no change in the rate of renal excretion. ELISA showed that serum soluble leptin receptor, known to antagonize leptin transport, had a 400‐fold increase, probably contributing to the hyperleptinemia and reduced tissue uptake. Thus, the ELKO mutation unexpectedly increased the stability of leptin but suppressed its tissue uptake. These changes probably contribute to the known partial resistance of the ELKO mice to diet‐induced obesity. J. Cell. Physiol. 228: 1610–1616, 2013. © 2013 Wiley Periodicals, Inc.     

Underlying the Mechanism of Vancomycin and Human Serum Albumin Interaction: A Biophysical Study

In the present study, the binding mechanism of vancomycin with human serum albumin (HSA) was determined. Upon addition of vancomycin to HSA, the fluorescence emission was quenched and the binding constant of vancomycin with HSA was found to be 6.05 × 10³ M^?1 at 295 K, which corresponds to –2.16 × 10⁴ J·mol^?1 of free energy. The conformation of HSA was altered upon binding of vancomycin with a decrease in α helix and an increase in β sheets and random coils, suggesting partial unfolding of the secondary structure. Molecular docking experiments found that vancomycin binds strongly with HSA at the hydrophobic pocket through hydrogen bonding and van der Waals interactions. An average binding distance of 4.71 nm has been determined on the basis of the Förster resonance energy theory. It was demonstrated that vancomycin binding to HSA causes protein structural changes. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:463‐470, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21511