Characterization of choroid plexus in the preterm rabbit pup following subcutaneous administration of recombinant human IGF-1/IGFBP-3.

Insulin-like growth factor-1 (IGF-1) is essential for normal brain development and regulates essential processes of vascular maturation and stabilization. Importantly, preterm birth is associated with reduced serum levels of IGF-1 as compared to in utero levels. Using a preterm rabbit pup model, we investigated the uptake of systemic recombinant human (rh) IGF-1 in complex with its main binding protein IGF-binding protein 3 (BP-3) to the brain parenchyma via the choroid plexus. Five hours after subcutaneous administration, labeled rhIGF-1/rhIGFBP-3 displayed a widespread presence in the choroid plexus of the lateral and third ventricle, however, to a less degree in the fourth, as well as in the perivascular and subarachnoid space. We found a time-dependent uptake of IGF-1 in cerebrospinal fluid, decreasing with postnatal age, and a translocation of IGF-1 through the choroid plexus. The impact of systemic rhIGF-1/rhIGFBP-3 on IGF-1 receptor activation in the choroid plexus decreased with postnatal age, correlating with IGF-1 uptake in cerebrospinal fluid. In addition, choroid plexus gene expression was observed to increase with postnatal age. Moreover, using choroid plexus in vitro cell cultures, gene expression and protein synthesis were further investigated upon rhIGF-1/rhIGFBP-3 stimulation as compared to rhIGF-1 alone, and found not to be differently altered. Here, we characterize the uptake of systemic rhIGF-1/rhIGFBP-3 to the preterm brain, and show that the interaction between systemic rhIGF-1/rhIGFBP-3 and choroid plexus varies over time.

Recombinant α1-Microglobulin (rA1M) Protects against Hematopoietic and Renal Toxicity, Alone and in Combination with Amino Acids, in a 177Lu-DOTATATE Mouse Radiation Model.

177Lu-DOTATATE peptide receptor radionuclide therapy (PRRT) is used clinically to treat metastasized or unresectable neuroendocrine tumors (NETs). Although 177Lu-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. α1-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 177Lu-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-177Lu-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 177Lu-DOTATATE, may reduce hematopoietic and kidney toxicity during PRRT with 177Lu-DOTATATE.

Targeting elevated heme levels to treat a mouse model for Diamond-Blackfan Anemia.

Diamond-Blackfan anemia (DBA) is a rare genetic disorder in which patients present a scarcity of erythroid precursors in an otherwise normocellular bone marrow. Most, but not all, patients carry mutations in ribosomal proteins such as RPS19, suggesting that compromised mRNA translation and ribosomal stress are pathogenic mechanisms causing depletion of erythroid precursors. To gain further insight to disease mechanisms in DBA, we performed a custom short hairpin RNA (shRNA) based screen against 750 genes hypothesized to affect DBA pathophysiology. Among the hits were two shRNAs against the erythroid specific heme-regulated eIF2α kinase (HRI), which is a negative regulator of mRNA translation. This study shows that shRNA-mediated HRI silencing or loss of one HRI allele improves expansion of Rps19-deficient erythroid precursors, as well as improves the anemic phenotype in Rps19-deficient animals. We found that Rps19-deficient erythroblasts have elevated levels of unbound intracellular heme, which is normalized by HRI heterozygosity. Additionally, targeting elevated heme levels by treating cells with the heme scavenger alpha-1-microglobulin (A1M), increased proliferation of Rps19-deficient erythroid precursors and decreased heme levels in a disease-specific manner. HRI heterozygosity, but not A1M treatment, also decreased the elevated p53 activity observed in Rps19-deficient cells, indicating that p53 activation is caused by ribosomal stress and aberrant mRNA translation and not heme overload in Rps19-deficiency. Together, these findings suggest that targeting elevated heme levels is a promising new treatment strategy for DBA.

177Lu-PSMA-617 Therapy in Mice, with or without the Antioxidant α1-Microglobulin (A1M), Including Kidney Damage Assessment Using 99mTc-MAG3 Imaging.

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.

Non-Invasive Imaging Methodologies for Assessment of Radiation Damage to Bone Marrow and Kidneys from Peptide Receptor Radionuclide Therapy.

BACKGROUND/AIMS: Peptide receptor radionuclide therapy (PRRT) is becoming clinical routine for management of neuroendocrine tumours. The number of PRRT cycles is correlated with treatment effect but theoretically limited by off-target radiation damage to kidneys and bone marrow. New imaging biomarkers for assessment of PRRT tissue damage would enable evaluation of novel renal and bone marrow protective agents, as well as personalised PRRT treatment regiments. METHODS: Mice treated with [177Lu]Lu-DOTA-TATE PRRT or vehicle were examined at baseline and following treatment with [18F]fluorothymidine (FLT) positron emission tomography (PET) and technetium-99m-mercapto-acetyl-tri-glycine ([99mTc]Tc-Mag3) single-photon emission tomography (SPECT) to assess dynamic changes in bone marrow proliferation and renal function, respectively. RESULTS: Bone marrow proliferation as assessed by [18F]FLT was decreased 2 days after PRRT treatment, but not vehicle, compared to baseline (target-to-background ratio [TBRmax] baseline:1.69 ± 0.29 vs. TBRmax PRRT: 0.91 ± 0.02, p < 0.01). Renal function as assessed by [99mTc]Tc-Mag3 SPECT was similarly decreased 2 days following PRRT compared to vehicle (fractional uptake rate [FUR] vehicle: 0.030 ± 0.014 s-1 vs. FUR PRRT: 0.0051 ± 0.0028 s-1, p < 0.01). CONCLUSION: [18F]FLT PET and [99mTc]Tc-Mag3 SPECT are promising techniques for assessing bone marrow and renal injury from [177Lu]Lu-DOTA-TATE PRRT and may potentially improve patient management by allowing evaluation of protective interventions as well as enabling personalised PRRT treatments.

The heme and radical scavenger α1-microglobulin (A1M) confers early protection of the immature brain following preterm intraventricular hemorrhage.

BACKGROUND: Germinal matrix intraventricular hemorrhage (GM-IVH) is associated with cerebro-cerebellar damage in very preterm infants, leading to neurodevelopmental impairment. Penetration, from the intraventricular space, of extravasated red blood cells and extracellular hemoglobin (Hb), to the periventricular parenchyma and the cerebellum has been shown to be causal in the development of brain injury following GM-IVH. Furthermore, the damage has been described to be associated with the cytotoxic nature of extracellular Hb-metabolites. To date, there is no therapy available to prevent infants from developing either hydrocephalus or serious neurological disability. Mechanisms previously described to cause brain damage following GM-IVH, i.e., oxidative stress and Hb-metabolite toxicity, suggest that the free radical and heme scavenger α1-microglobulin (A1M) may constitute a potential neuroprotective intervention. METHODS: Using a preterm rabbit pup model of IVH, where IVH was induced shortly after birth in pups delivered by cesarean section at E29 (3 days prior to term), we investigated the brain distribution of recombinant A1M (rA1M) following intracerebroventricular (i.c.v.) administration at 24 h post-IVH induction. Further, short-term functional protection of i.c.v.-administered human A1M (hA1M) following IVH in the preterm rabbit pup model was evaluated. RESULTS: Following i.c.v. administration, rA1M was distributed in periventricular white matter regions, throughout the fore- and midbrain and extending to the cerebellum. The regional distribution of rA1M was accompanied by a high co-existence of positive staining for extracellular Hb. Administration of i.c.v.-injected hA1M was associated with decreased structural tissue and mitochondrial damage and with reduced mRNA expression for proinflammatory and inflammatory signaling-related genes induced by IVH in periventricular brain tissue. CONCLUSIONS: The results of this study indicate that rA1M/hA1M is a potential candidate for neuroprotective treatment following preterm IVH.

Midsummer Eve in Sweden: a natural fluid challenge in patients with heart failure.

AIMS: Volume overload exacerbations in patients with chronic heart failure (HF) are associated with a dismal prognosis and are often triggered by dietary incompliance. We aimed to describe the effects of dietary changes on measures of fluid retention in HF patients. METHODS AND RESULTS: Thirty-one HF patients with an implantable defibrillator (age 64 ± 11 years, ejection fraction 25 ± 12%, median NT-proBNP 2090 ng/L, resynchronization therapy: n= 25) were followed by daily body weight (BW) (telemonitoring) and intrathoracic impedance (by the implanted device) around Swedish Midsummer 2009, a holiday traditionally celebrated with meals including salty fish dishes and ample intake of fluids. Midsummer Eve celebrations caused a distinct and rapid increase in BW and a decrease in impedance indicating increased fluid retention. Compared with baseline values, peak BW increased by 1225 g [interquartile range (IQR) 475-2013 g)] which was accompanied by a decrease in impedance (3 Ω; IQR -5.2 to -1.2) and a clear deflection on the impedance-based fluid detection algorithm (OptiVol) that crossed the preset fluid-alert threshold six times following Midsummer. Body weight and impedance values were normalized after a period of 20 and 8 days, respectively. A clustering of minor clinical events following Midsummer suggests a possible adverse impact of dietary incompliance. However, none of the patients were hospitalized for HF. CONCLUSIONS: Dietary incompliance, e.g. on the occasion of a ceremonial meal, may lead to marked disturbances in the fluid balance of patients with HF reflected by increased BW and decreased intrathoracic impedance. These findings underline the importance of maintaining stable volume conditions in HF patients.

Three year outcome of cardiac resynchronization therapy: a single center evaluation.

AIMS: To evaluate the long-term clinical outcome and device performance of cardiac resynchronization therapy in a consecutive sample of patients with moderate to severe heart failure. METHODS AND RESULTS: Between 1998 and 2000, forty consecutive patients with drug-refractory heart failure due to ischemic or idiopathic dilated cardiomyopathy were selected for cardiac resynchronization therapy (CRT). After successful implantation of the coronary sinus lead (n = 35, 88%), patients were followed every sixth month by New York Heart Association (NYHA) functional class, the 6-minute walking test (6 walk), quality of life (QoL, Minnesota), and pacemaker control. NYHA-class and 6 walk were significantly improved after 6 months and continued to improve gradually until 36 months of follow-up. The QoL improvement at 6 months was sustained over 3 years. After 3 years, the beta-blocker dose could be increased in 10/23 patients as compared to baseline. Nine patients had to be re-operated. Coronary sinus lead thresholds were stable over time. CONCLUSION: The clinical improvements by CRT are sustained over 3 years of follow-up. In the setting of a University Medical Center, CRT can be applied in clinical routine with excellent clinical outcome, acceptable implantation success, and stable device performance over time.