| 1. |
- Alattar, Abdul Ghani, et al.
(författare)
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Recombinant alpha(1)-Microglobulin (rA1M) Protects against Hematopoietic and Renal Toxicity, Alone and in Combination with Amino Acids, in a Lu-177-DOTATATE Mouse Radiation Model
- 2023
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Ingår i: Biomolecules. - 2218-273X. ; 13:6
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Tidskriftsartikel (refereegranskat)abstract
- Lu-177-DOTATATE peptide receptor radionuclide therapy (PRRT) is used clinically to treat metastasized or unresectable neuroendocrine tumors (NETs). Although Lu-177-DOTATATE is mostly well tolerated in patients, bone marrow suppression and long-term renal toxicity are still side effects that should be considered. Amino acids are often used to minimize renal radiotoxicity, however, they are associated with nausea and vomiting in patients. alpha (1)-microglobulin (A1M) is an antioxidant with heme- and radical-scavenging abilities. A recombinant form (rA1M) has previously been shown to be renoprotective in preclinical models, including in PRRT-induced kidney damage. Here, we further investigated rA1M's renal protective effect in a mouse Lu-177-DOTATATE model in terms of administration route and dosing regimen and as a combined therapy with amino acids (Vamin). Moreover, we investigated the protective effect of rA1M on peripheral blood and bone marrow cells, as well as circulatory biomarkers. Intravenous (i.v.) administration of rA1M reduced albuminuria levels and circulatory levels of the oxidative stress-related protein fibroblast growth factor-21 (FGF-21). Dual injections of rA1M (i.e., at 0 and 24 h post-Lu-177-DOTATATE administration) preserved bone marrow cellularity and peripheral blood reticulocytes. Administration of Vamin, alone or in combination with rA1M, did not show any protection of bone marrow cellularity or peripheral reticulocytes. In conclusion, this study suggests that rA1M, administered i.v. for two consecutive days in conjunction with Lu-177-DOTATATE, may reduce hematopoietic and kidney toxicity during PRRT with Lu-177-DOTATATE.
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| 2. |
- Bergwik, Jesper, et al.
(författare)
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Binding of the human antioxidation protein α1-microglobulin (A1M) to heparin and heparan sulfate. Mapping of binding site, molecular and functional characterization, and co-localization in vivo and in vitro
- 2021
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Ingår i: Redox Biology. - : Elsevier BV. - 2213-2317. ; 41
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Tidskriftsartikel (refereegranskat)abstract
- Heparin and heparan sulfate (HS) are linear sulfated disaccharide polymers. Heparin is found mainly in mast cells, while heparan sulfate is found in connective tissue, extracellular matrix and on cell membranes in most tissues. α1-microglobulin (A1M) is a ubiquitous protein with thiol-dependent antioxidant properties, protecting cells and matrix against oxidative damage due to its reductase activities and radical- and heme-binding properties. In this work, it was shown that A1M binds to heparin and HS and can be purified from human plasma by heparin affinity chromatography and size exclusion chromatography. The binding strength is inversely dependent of salt concentration and proportional to the degree of sulfation of heparin and HS. Potential heparin binding sites, located on the outside of the barrel-shaped A1M molecule, were determined using hydrogen deuterium exchange mass spectrometry (HDX-MS). Immunostaining of endothelial cells revealed pericellular co-localization of A1M and HS and the staining of A1M was almost completely abolished after treatment with heparinase. A1M and HS were also found to be co-localized in vivo in the lungs, aorta, kidneys and skin of mice. The redox-active thiol group of A1M was unaffected by the binding to HS, and the cell protection and heme-binding abilities of A1M were slightly affected. The discovery of the binding of A1M to heparin and HS provides new insights into the biological role of A1M and represents the basis for a novel method for purification of A1M from plasma.
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| 3. |
- Bergwik, Jesper, et al.
(författare)
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Knockout of the radical scavenger α1-microglobulin in mice results in defective bikunin synthesis, endoplasmic reticulum stress and increased body weight
- 2021
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Ingår i: Free Radical Biology and Medicine. - : Elsevier BV. - 0891-5849. ; 162
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Tidskriftsartikel (refereegranskat)abstract
- α1-microglobulin (A1M) is a ubiquitous protein with reductase and radical- and heme-binding properties. The protein is mainly expressed in the liver and encoded by the α1-microglobulin-bikunin precursor (AMBP) gene together with the plasma proteinase inhibitor bikunin. The AMBP polypeptide is translated, glycosylated and the C-terminal bikunin part linked via a chondroitin sulfate glycosaminoglycan chain to one or two heavy chains in the endoplasmic reticulum (ER) and Golgi compartments. After proteolytic cleavage, the A1M protein and complexed bikunin parts are secreted separately. The complete physiological role of A1M, and the reason for the co-synthesis with bikunin, are both still unknown. The aim of this work was to develop an A1M knockout (A1M−KO) mouse model lacking expression of A1M, but with a preserved bikunin expression, and to study the phenotypic traits in these mice, with a focus on hepatic endoplasmic reticulum (ER) function. The bikunin expression was increased in the A1M−KO mouse livers, while the bikunin levels in plasma were decreased, indicating a defective biosynthesis of bikunin. The A1M−KO livers also showed an increased expression of transducers of the unfolded protein response (UPR), indicating an increased ER-stress in the livers. At twelve months of age, the A1M−KO mice also displayed an increased body weight, and an increased liver weight and lipid accumulation. Moreover, the KO mice showed an increased expression of endogenous antioxidants in the liver, but not in the kidneys. Together, these results suggest a physiological role of A1M as a regulator of the intracellular redox environment and more specifically the ER folding and posttranslational modification processes, particularly in the liver.
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| 4. |
- Bergwik, Jesper, et al.
(författare)
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Structure, Functions, and Physiological Roles of the Lipocalin α1-Microglobulin (A1M)
- 2021
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Ingår i: Frontiers in Physiology. - : Frontiers Media SA. - 1664-042X. ; 12
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Forskningsöversikt (refereegranskat)abstract
- α1-microglobulin (A1M) is found in all vertebrates including humans. A1M was, together with retinol-binding protein and β-lactoglobulin, one of the three original lipocalins when the family first was proposed in 1985. A1M is described as an antioxidant and tissue cleaning protein with reductase, heme- and radical-binding activities. These biochemical properties are driven by a strongly electronegative surface-exposed thiol group, C34, on loop 1 of the open end of the lipocalin barrel. A1M has been shown to have protective effects in vitro and in vivo in cell-, organ-, and animal models of oxidative stress-related medical conditions. The gene coding for A1M is unique among lipocalins since it is flanked downstream by four exons coding for another non-lipocalin protein, bikunin, and is consequently named α1-microglobulin-bikunin precursor gene (AMBP). The precursor is cleaved in the Golgi, and A1M and bikunin are secreted from the cell separately. Recent publications have suggested novel physiological roles of A1M in regulation of endoplasmic reticulum activities and erythrocyte homeostasis. This review summarizes the present knowledge of the structure and functions of the lipocalin A1M and presents a current model of its biological role(s).
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| 5. |
- Carlsson, Magnus L.R., et al.
(författare)
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Expression, Purification and Initial Characterization of Functional α1-Microglobulin (A1M) in Nicotiana benthamiana
- 2020
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Ingår i: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- α1-Microglobulin (A1M) is a small glycoprotein that belongs to the lipocalin protein family. A major biological role of A1M is to protect cells and tissues against oxidative damage by clearing free heme and reactive oxygen species. Because of this, the protein has attracted great interest as a potential pharmaceutical candidate for treatment of acute kidney injury and preeclampsia. The aim of this study was to explore the possibility of expressing human A1M in plants through transient gene expression, as an alternative or complement to other expression systems. E. coli, insect and mammalian cell culture have previously been used for recombinant A1M (rA1M) or A1M production, but these systems have various drawbacks, including additional complication and expense in refolding for E. coli, while insect produced rA1M is heavily modified with chromophores and mammalian cell culture has been used only in analytical scale. For that purpose, we have used a viral vector (pJL-TRBO) delivered by Agrobacterium for expression of three modified A1M gene variants in the leaves of N. benthamiana. The results showed that these modified rA1M protein variants, A1M-NB1, A1M-NB2 and A1M-NB3, targeted to the cytosol, ER and extracellular space, respectively, were successfully expressed in the leaves, which was confirmed by SDS-PAGE and Western blot analysis. The cytosol accumulated A1M-NB1 was selected for further analysis, as it appeared to have a higher yield than the other variants, and was purified with a yield of ca. 50 mg/kg leaf. The purified protein had the expected structural and functional properties, displaying heme-binding capacity and capacity of protecting red blood cells against stress-induced cell death. The protein also carried bound chromophores, a characteristic feature of A1M and an indicator of a capacity to bind small molecules. The study showed that expression of the functional protein in N. benthamiana may be an attractive alternative for production of rA1M for pharmaceutical purposes and a basis for future research on A1M structure and function.
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| 6. |
- Ekström, Claes, et al.
(författare)
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Evaluation of recombinant human IGF-1/IGFBP-3 on intraventricular hemorrhage prevention and survival in the preterm rabbit pup model
- 2023
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Ingår i: Scientific Reports. - 2045-2322. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Insulin-like growth factor-1 (IGF-1) is essential for normal brain development and regulates processes of vascular maturation. The pathogenesis of intraventricular hemorrhage (IVH) relates to the fragility of the immature capillaries in the germinal matrix, and its inability to resist fluctuations in cerebral blood flow. In this work, using different experimental setups, we aimed to (i) establish an optimal time-point for glycerol-induction of IVH in relation to time-point of recombinant human (rh) IGF-1/rhIGFBP-3 administration, and (ii) to evaluate the effects of a physiologic replacement dose of rhIGF-1/rhIGFBP-3 on prevention of IVH and survival in the preterm rabbit pup. The presence of IVH was evaluated using high-frequency ultrasound and post-mortem examinations. In the first part of the study, the highest incidence of IVH (> 60%), occurred when glycerol was administered at the earliest timepoint, e.g., 6h after birth. At later time-points (18 and 24h) the incidence decreased substantially. In the second part of the study, the incidence of IVH and mortality rate following rhIGF-1/rhIGFBP-3 administration was not statistically different compared to vehicle treated animals. To evaluate the importance of maintaining intrauterine serum levels of IGF-1 following preterm birth, as reported in human interventional studies, additional studies are needed to further characterize and establish the potential of rhIGF-1/rhIGFBP-3 in reducing the prevalence of IVH and improving survival in the preterm rabbit pup.
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| 7. |
- Kristiansson, Amanda, et al.
(författare)
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177Lu-PSMA-617 Therapy in Mice, with or without the Antioxidant α1-Microglobulin (A1M), Including Kidney Damage Assessment Using 99mTc-MAG3 Imaging
- 2021
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Ingår i: Biomolecules. - : MDPI AG. - 2218-273X. ; 11:2
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Tidskriftsartikel (refereegranskat)abstract
- Anti-prostate specific membrane antigen (PSMA) radioligand therapy is promising but not curative in castration resistant prostate cancer. One way to broaden the therapeutic index could be to administer higher doses in combination with radioprotectors, since administered radioactivity is kept low today in order to avoid side-effects from a high absorbed dose to healthy tissue. Here, we investigated the human radical scavenger α1-microglobulin (A1M) together with 177-Lutetium (177Lu) labeled PSMA-617 in preclinical models with respect to therapeutic efficacy and kidney toxicity. Nude mice with subcutaneous LNCaP xenografts were injected with 50 or 100 MBq of [177Lu]Lu-PSMA-617, with or without injections of recombinant A1M (rA1M) (at T = 0 and T = 24 h). Kidney absorbed dose was calculated to 7.36 Gy at 4 days post a 100 MBq injection. Activity distribution was imaged with Single-Photon Emission Computed Tomography (SPECT) at 24 h. Tumor volumes were measured continuously, and kidneys and blood were collected at termination (3-4 days and 3-4 weeks after injections). In a parallel set of experiments, mice were given [177Lu]Lu-PSMA-617 and rA1M as above and dynamic technetium-99m mercaptoacetyltriglycine ([99mTc]Tc-MAG3) SPECT imaging was performed prior to injection, and 3- and 6-months post injection. Blood and urine were continuously sampled. At termination (6 months) the kidneys were resected. Biomarkers of kidney function, expression of stress genes and kidney histopathology were analyzed. [177Lu]Lu-PSMA-617 uptake, in tumors and kidneys, as well as treatment efficacy did not differ between rA1M and vehicle groups. In mice given rA1M, [99mTc]Tc-MAG3 imaging revealed a significantly higher slope of initial uptake at three months compared to mice co-injected with [177Lu]Lu-PSMA-617 and vehicle. Little or no change compared to control was seen in urine albumin, serum/plasma urea levels, RT-qPCR analysis of stress response genes and in the kidney histopathological evaluation. In conclusion, [99mTc]Tc-MAG3 imaging presented itself as a sensitive tool to detect changes in kidney function revealing that administration of rA1M has a potentially positive effect on kidney perfusion and tubular function when combined with [177Lu]Lu-PSMA-617 therapy. Furthermore, we could show that rA1M did not affect anti-PSMA radioligand therapy efficacy.
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| 8. |
- Kristiansson, Amanda, et al.
(författare)
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Hematological and renal toxicity in mice after three cycles of high activity [177Lu]Lu-PSMA-617 with or without human α1-microglobulin
- 2024
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Ingår i: Scientific Reports. - 2045-2322. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Radioligand therapy with [177Lu]Lu-PSMA-617 can be used to prolong life and reduce tumor burden in terminally ill castration resistant prostate cancer patients. Still, accumulation in healthy tissue limits the activity that can be administered. Therefore, fractionated therapy is used to lower toxicity. However, there might be a need to reduce toxicity even further with e.g. radioprotectors. The aim of this study was to (i). establish a preclinical mouse model with fractionated high activity therapy of three consecutive doses of 200 MBq [177Lu]Lu-PSMA-617 in which we aimed to (ii). achieve measurable hematotoxicity and nephrotoxicity and to (iii). analyze the potential protective effect of co-injecting recombinant α1-microglobulin (rA1M), a human antioxidant previously shown to have radioprotective effects. In both groups, three cycles resulted in increased albuminuria for each cycle, with large individual variation. Another marker of kidney injury, serum blood urea nitrogen (BUN), was only significantly increased compared to control animals after the third cycle. The number of white and red blood cells decreased significantly and did not reach the levels of control animals during the experiment. rA1M did reduce absorbed dose to kidney but did not show significant protection here, but future studies are warranted due to the recent clinical studies showing a significant renoprotective effect in patients.
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| 9. |
- Kristiansson, Amanda, et al.
(författare)
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Hematological Toxicity in Mice after High Activity Injections of 177Lu-PSMA-617
- 2022
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Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 14:4
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Tidskriftsartikel (refereegranskat)abstract
- Prostate cancer (PC) is one of the most common malignancies affecting men, with poor prognosis after progression to metastatic castration-resistant prostate cancer (mCRPC). Radioligand therapy (RLT) targeting the overexpressed PSMA on PC cells, with, e.g., 177Lu-PSMA-617, has been effective in reducing tumor burden and prolonging survival in mCRPC. However, it is not a curative method with kidney and bone marrow toxicity limiting the activity given to patients. Previous preclinical models have reported transient hematotoxicity for up to 120 MBq. This activity may still be too low to investigate the effect on renal function since it corresponds to an absorbed dose below 10 Gy, whereas the kidneys in a clinical setting usually receive an absorbed dose more than double. Here we investigated the hematotoxicity and recovery after administered activities of 120, 160, and 200 MBq in a 177Lu-PSMA-617 BALB/cAnNRj mouse model. The animals had an initial drop in white blood cells (WBC) starting 4 days post injection, which recovered after 21 days. The effect on red blood cells (RBC) and platelets was detected later; 17 days post-injection levels decreased compared to the control group. The reduction was restored again 32 days post injection. No correlation between injected activity and hematotoxicity was found. Our results suggest that activities up to 200 MBq of 177Lu-PSMA-617 give transient hematotoxicity from which animals recover within a month and no radiation-related deaths. Injecting these high activities could allow animal studies with increased clinical relevance when studying renal toxicity in animal models.
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| 10. |
- Kristiansson, Amanda, et al.
(författare)
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Human radical scavenger α1-microglobulin protects against hemolysis in vitro and α1-microglobulin knockout mice exhibit a macrocytic anemia phenotype
- 2021
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Ingår i: Free Radical Biology & Medicine. - : Elsevier BV. - 0891-5849. ; 162
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Tidskriftsartikel (refereegranskat)abstract
- During red blood cell (RBC) lysis hemoglobin and heme leak out of the cells and cause damage to the endothelium and nearby tissue. Protective mechanisms exist; however, these systems are not sufficient in diseases with increased extravascular hemolysis e.g. hemolytic anemia. α1-microglobulin (A1M) is a ubiquitous reductase and radical- and heme-binding protein with antioxidation properties. Although present in the circulation in micromolar concentrations, its function in blood is unclear. Here, we show that A1M provides RBC stability. A1M-/- mice display abnormal RBC morphology, reminiscent of macrocytic anemia conditions, i.e. fewer, larger and more heterogeneous cells. Recombinant human A1M (rA1M) reduced in vitro hemolysis of murine RBC against spontaneous, osmotic and heme-induced stress. Moreover, A1M is taken up by human RBCs both in vitro and in vivo. Similarly, rA1M also protected human RBCs against in vitro spontaneous, osmotic, heme- and radical-induced hemolysis as shown by significantly reduced leakage of hemoglobin and LDH. Addition of rA1M resulted in decreased hemolysis compared to addition of the heme-binding protein hemopexin and the radical-scavenging and reducing agents ascorbic acid and Trolox (vitamin E). Furthermore, rA1M significantly reduced spontaneous and heme-induced fetal RBC cell death. Addition of A1M to human whole blood resulted in a significant reduction of hemolysis, whereas removal of A1M from whole blood resulted in increased hemolysis. We conclude that A1M has a protective function in reducing hemolysis which is neither specific to the origin of hemolytic insult, nor species specific.
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