Lee, T. et al (1981) proposed the encapsulation of hormones such as progesterone into serum albumin beads, such that their in vivo proteolysis would allow a gradual release of hormone at low levels, for extended hormone action. It was proposed, in the Department of Microbiology, Rhodes University, to replace the hormone component of the above bead formulation, with virus as antigen, in the development of a vaccine. Beads optimally crosslinked at 1% final glutaraldehyde concentration, containing Nodamura virus, were shown to promote an adjuvant effect in vivo, analogous to the release of antigen from Freund's Complete Adjuvant (FCA), so that extended immunostimulation resulted. It was shown that soluble antigen promoted a short-lived primary immune response, peaking around day 25 following inoculation. Antigen presented in beads, on the other hand, initially elicited a low humoral response, but this response gradually increased up to a peak around day 110 post inoculation, before decreasing. No apparent adverse side-effects were noted following inoculation of antigen-containing serum albumin beads, compared to necrosis following antigen in FCA inoculation, supporting the proposal of using albumin homotypic for the test inoculee animal, so that the beads would themselves be non-immunogenic and would merely act as a vehicle in the vaccine formulation. The indirect enzyme-linked immunosorbent assay (ELISA) was used to monitor the humoral response to antigen following inoculation. Results showed that covalent crosslinking of albumin in the formation of the beads did not promote immunogenicity on the part of the chemically altered albumin. The ELISA test was used to indicate the kinetics of the IgG response to Nodamura virus when presented in formulations such as: Freely soluble virus or its subunit; soluble intact virus inactivated by treatment with glutaraldehyde; intact virus entrapped in serum albumin beads cross; linked at different percentage final glutaraldehyde concentrations and also virus subunit prepared in albumin beads. The presence of virus-neutral ising antibodies was noted in serum obtained from rabbits inoculated with virus entrapped in albumin beads. Virus infectivity, titrated in mice, showed protection against virus challenge after incubation of virus with serum obtained above.

Plasma kallikrein (PK) is a coagulation factor that activates Factor XII in the contact pathway and generates vasoactive bradykinin from kininogen. C1-esterase inhibitor (C1INH) regulates PK levels. C1INH deficiency manifests as potentially life-threatening hereditary angioedema (HAE). Several drugs are licensed for HAE treatment and/or prophylaxis, including C1INH concentrates and recombinant Ecallantide (rEcall). rEcall is a small protein comprised of the first Kunitz domain of Tissue Factor Pathway Inhibitor and was substituted at seven residues making it a PK-specific inhibitor. rEcall is licensed for HAE treatment, but its short half-life prevents prophylactic application. This project focused on comparing the inhibition of PK and the in vivo clearance of hexahistidine-tagged rEcall (H6-rEcall) and H6-rEcall genetically fused to human serum albumin (H6-rEcall-HSA), or an albumin binding domain (H6-rEcall-ABD). To determine if the orientation of the constructs contributed to activity and half-life, proteins were reoriented, and HSA was moved from the C- to N-terminus. Proteins produced were H6-rEcall (64 amino acids), H6-rEcall-ABD (110 amino acids), H6-rEcall-HSA (666 amino acids), H6-rEcall-HSA, rEcall-H6 (64 amino acids), and HSA-rEcall- H6 (666 amino acids). All proteins were expressed in Pichia pastoris, secreted, and purified from conditioned media via nickel-chelate chromatography. PK activity was assessed via amidolysis of S2302, and the inhibitory constant (Ki) was determined. Purified proteins were radiolabeled with 125I and injected intravenously into CD1 mice. Acid-precipitable radioactivity in timed blood samples was expressed as a percentage of the peak post-injection value. Values were means ± SD, n = 6. Next, the role of Lipoprotein related receptor protein 1 (LRPI) or neonatal Fc receptor (FcRn) in rEcall proteins’ clearance was examined by performing a competition experiment. In this experiment, rEcall associated with albumin was co-administered with a competitor ligand that blocked FcRn or LRP1. To further investigate the role of these receptors and the organs responsible for rEcall proteins’ clearance, organ distribution was conducted by cannulating mice via the right jugular vein and delivering 125I-rEcall or 125I-HSA-rEcall. The organs analyzed were the kidneys, liver, spleen, and heart. In vitro, rEcall and its modified proteins had a Ki of ~ 2 – 3 nM. In vivo, fusion proteins behaved similarly and presented some pharmacokinetic enhancements. Two hours post-injection, 50 ± 10% of H6-rEcall-HSA and 36 ± 6 % of HSA-rEcall-H6 remained in the circulation vs. 6 ± 2 % of H6-rEcall and 7 ± 1% of H6-rEcall-ABD (p <0.0001), while 75 ± 5% of HSA-H6 was recovered in the circulation. In the competition experiment, the presence of GST-RAP elevated the recovery of 125I- H6-rEcall-HSA 2.2-fold, from 37 ± 3 % for GST + H6-rEcall-HSA to 81 ± 3 % for GST-RAP + H6-rEcall-HSA. Competition with IVIg also significantly increased the recovery of 125I- H6- rEcall-HSA, but to a lesser degree, by 1.13-fold (to 42 ± 5% for IVIg + H6-rEcall-HSA). Similar significant changes were also observed 30 mins after injection, of 2.9- and 1.9-fold, respectively. Finally, results indicate that albumin changed rEcall’s organ distribution. Of the four organs extracted 30 minutes after injection, rEcall-H6 was predominantly found in the kidneys, and fusion to albumin largely redirected rEcall to the liver. 3.6-fold more HSA-rEcall-H6 was found in the liver in comparison to rEcall (p<0.0001). In contrast, 3.3-fold more rEcall was observed in the kidneys vs. rEcall-HSA (p<0.0001). In conclusion, fusing HSA to either the N- or C-terminus of rEcall extended its plasma residency time and did not impact its function towards PK. / Thesis / Master of Health Sciences (MSc)

No description available.

Objective. Albumin is an abundant and slowly-cleared plasma protein. Our laboratory previously incorporated albumin into recombinant fusion proteins to extend the plasma half-life of small proteins of potential therapeutic utility. We sought to determine if reiteration of albumin in a single polypeptide chain would further extend its half-life. Design. Hexahistidine-tagged rabbit serum albumin (RSA) with Cys 34 altered to Ala to prevent disulphide-bonded dimerization was produced in yeast (H₆-RSA-C34A), and compared to a reiterated forma of H₆-RSA-C34A containing two domains of amino acids 1-584 separated by a hexaglycine spacer. Clearance of these purified iodinated proteins was compared in rabbits. Materials and Methods. Site-directed mutagenesis employing PCR was used to alter the encoding plasmid. Proteins secreted from transformed Pichia pastoris yeast cell lines were purified using nickel-chelated affinity chromatography and radioiodinated by the Iodogen method. Labeled proteins were injected intravenously into rabbits and the residual acid-precipitable protein concentration in serial plasma samples was determined over time. Results. P. 𝘱𝘢𝘴𝘵𝘰𝘳𝘪𝘴 cells transformed with the expression plasmids secreted 140 kDa H₆-diRSA and 70 kDa H₆-RSA-C34A proteins, which were purified to apparent homogeneity. Mean terminal catabolic half-lives (±SD) were 4.9 (±0.7) and 3.0 (±0.3) days for H₆-RSA-C34A and H₆-diRSA, respectively. Then values were 9 for H₆-diRSA and 12 for H₆-RSA-C34A. conclusions. H₆-diRSA was cleared from the circulation more rapidly than H₆RSA-C34A. We hypothesized that increased catabolism of the reiterated molecule could be due to an increased rate of cellular uptake and endocytosis of H₆-diRSA due to increased avidity for cellular binding sites. Non-reiterated albumin therefore appears to be optimal as a carrier protein for small recombinant blood products. / Thesis / Master of Science (MSc)

abstract: Exposure of blood plasma/serum (P/S) to thawed conditions, greater than -30°C, can produce biomolecular changes that misleadingly impact measurements of clinical markers within archived samples. Reported here is a low sample-volume, dilute-and-shoot, intact protein mass spectrometric assay of albumin proteoforms called “ΔS-Cys-Albumin” that quantifies cumulative exposure of archived P/S samples to thawed conditions. The assay uses the fact that S-cysteinylation (oxidation) of albumin in P/S increases to a maximum value when exposed to temperatures greater than -30°C. The multi-reaction rate law that governs this albumin S-cysteinylation formation in P/S was determined and was shown to predict the rate of formation of S-cysteinylated albumin in P/S samples—a step that enables back-calculation of the time at which unknown P/S specimens have been exposed to room temperature. To emphasize the capability of this assay, a blind challenge demonstrated the ability of ΔS-Cys-Albumin to detect exposure of individual and grouped P/S samples to unfavorable storage conditions. The assay was also capable of detecting an anomaly in a case study of nominally pristine serum samples collected under NIH-sponsorship, demonstrating that empirical evidence is required to guarantee accurate knowledge of archived P/S biospecimen storage history. The ex vivo glycation of human serum albumin was also investigated showing that P/S samples stored above their freezing point leads to significant increases in glycated albumin. These increases were found to occur within hours at room temperature, and within days at -20 °C. These increases continued over a period of 1-2 weeks at room temperature and over 200 days at -20 °C, ultimately resulting in a doubling of glycated albumin in both healthy and diabetic patients. It was also shown that samples stored at lower surface area-to-volume ratios or incubated under a nitrogen atmosphere experienced less rapid glucose adduction of albumin—suggesting a role for oxidative glycation in the ex vivo glycation of albumin. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2018

No description available.

In clinical findings, complications are the major cause of death in cirrhotic patients. Among all the complications, ascites is most frequent type. All cirrhotic patients with ascites could develop spontaneous bacterial peritonitis (SBP). The hospitalized prevalence of SBP was high (30%) in cirrhotic ascites patients. However, the outcome of cirrhotic SBP patients has been improved because of using the antibiotics cephalosporins. Furthermore, treatment with intravenous albumin in addition to cephalosporins reduced the incidence of renal impairment and the mortality in SBP patients in a multicentre study. However, other studies showed that administration of albumin might not work as effectively as other plasma expanders do. Moreover, administration of large volume albumin would make the medication very expensive and might have potential risk of infectious disease, since the therapeutic albumin is extracted from human plasma. Therefore, to treat patients with large volume albumin become disputable. In this study, we evaluated the effects of small volume intravenous albumin with cephalosporins treatments through monitoring patients¡¦ renal and hepatic functions and related inflammatory markers. Results showed that plasma TNF-£\, £LL-6 and ascites endotoxin, TNF-£\ and IL-6 levels were significantly reduced in patients treated with cephalosporins plus small volume albumin, but not in those treated with cephalosporins alone. Also, the combination therapy of cephalosporins and small volume albumin avoid the dramatically elevation of plasma and ascites nitric oxide, as well as the further degree of renal function impairment. The positive results of this study laid a solid foundation for a large scale investigation.

No description available.

Thesis--Copenhagen. / Summary in Danish. Includes bibliographical references (p. 175-191).

Thesis--Copenhagen. / Summary in Danish. Bibliography: p. 175-191.