Transfusion of female blood in a rat model is associated with red blood cells entrapment in organs.

Transfusion of red blood cells (RBCs) has been associated with adverse outcomes. Mechanisms may be related to donor sex and biological age of RBC. This study hypothesized that receipt of female blood is associated with decreased post-transfusion recovery (PTR) and a concomitant increased organ entrapment in rats, related to young age of donor RBCs. Donor rats underwent bloodletting to stimulate production of new, young RBCs, followed by Percoll fractionation for further enrichment of young RBCs based on their low density. Control donors did not undergo these procedures. Male rats received either a (biotinylated) standard RBC product or a product enriched for young RBCs, derived from either male or female donors. Controls received saline. Organs and blood samples were harvested after 24 hours. This study found no difference in PTR between groups, although only the group receiving young RBCs from females failed to reach a PTR of 75%. Receipt of both standard RBCs and young RBCs from females was associated with increased entrapment of donor RBCs in the lung, liver, and spleen compared to receiving blood from male donors. Soluble ICAM-1 and markers of hemolysis were higher in recipients of female blood compared to control. In conclusion, transfusing RBCs from female donors, but not from male donors, is associated with trapping of donor RBCs in organs, accompanied by endothelial activation and hemolysis.

Can volume-reduced plasma products prevent transfusion-associated circulatory overload in a two-hit animal model?

BACKGROUND AND OBJECTIVES: Transfusion-associated circulatory overload (TACO) is a pulmonary transfusion complication and a leading cause of transfusion-related morbidity and mortality. Volume overload and rising hydrostatic pressure as a consequence of transfusion are seen as the central pathway leading to TACO. A possible preventative measure for TACO could be the use of low-volume blood products like volume-reduced lyophilized plasma. We hypothesize that volume-reduced lyophilized plasma decreases circulatory overload leading to a reduced pulmonary capillary pressure and can therefore be an effective strategy to prevent TACO. MATERIALS AND METHODS: A validated two-hit animal model in rats with heart failure was used. Animals were randomized to receive 4 units of either solvent-detergent pooled plasma (SDP) as control, standard volume lyophilized plasma (LP-S) or hyperoncotic volume-reduced lyophilized plasma (LP-VR). The primary outcome was the difference between pre-transfusion and post-transfusion left ventricular end-diastolic pressure (ΔLVEDP). Secondary outcomes included markers for acute lung injury. RESULTS: LVEDP increased in all randomization groups following transfusion. The greatest elevation was seen in the group receiving LP-VR (+11.9 mmHg [5.9-15.6]), but there were no significant differences when compared to groups receiving either LP-S (+6.3 mmHg [2.9-13.4], p = 0.29) or SDP (+7.7 mmHg [4.5-10.5], p = 0.55). There were no significant differences in markers for acute lung injury, such as pulmonary wet/dry weight ratios, lung histopathology scores or PaO2 /FiO2 ratio between the three groups. CONCLUSION: Transfusion with hyperoncotic volume-reduced plasma did not attenuate circulatory overload compared to standard volume plasma and was therefore not an effective preventative strategy for TACO in this rat model.

The effect of plasma transfusion in an experimental two-hit animal model of transfusion-associated circulatory overload with heart failure.

BACKGROUND: Transfusion-associated circulatory overload (TACO) is a leading cause of transfusion-related morbidity and mortality. TACO follows a two-hit pathophysiology, where comorbidities like cardiac or renal failure act as the first hit followed by blood transfusion as a second hit. Observational studies suggest that plasma transfusion is more likely to cause TACO than other blood products. We conducted a randomized animal study to gather evidence that plasma transfusion can induce TACO. MATERIAL AND METHODS: As a first hit a large myocardial infarction was created in male Wistar rats. Then animals were randomized to receive 4 units of solvent/ detergent-treated pooled plasma (SDP), fresh frozen plasma (FFP), a colloid control (albumin 5%) or a crystalloid fluid control (Ringer's lactate) (n=10 per group). The primary outcome was the difference between pre- and post-transfusion left-ventricular end diastolic pressure (ΔLVEDP). Secondary outcomes were markers for acute lung injury; lung wet/dry weight ratio, PaO2/FiO2 ratio and pulmonary histological assessment. RESULTS: Pre-transfusion characteristics were similar between groups. ΔLVEDP increased significantly after transfusion with SDP (7.7 mmHg; 4.5-10.5) and albumin (13.0 mmHg; 6.5-15.2), but not after FFP (7.9 mmHg, 1.1; 11.3) compared to infusion with Ringer's lactate (0.6 mmHg; 0.4-2.2), p=0.007, p=0.0005 and p=0.14 respectively. There were no significant differences in ΔLVEDP between groups receiving SDP, FFP or albumin. There was no increase in acute lung injury in any group compared to other groups. DISCUSSION: Circulatory overload, measured as ΔLVEDP, was induced after transfusion of SDP or albumin, but not after infusion of Ringer's lactate. These results show that the effect of plasma transfusion on ΔLVEDP differs from fluid overload induced by crystalloid infusion. Colloid osmotic pressure may be an important component in the development of TACO and should be a target for future research.

Aged versus fresh autologous platelet transfusion in a two-hit healthy volunteer model of transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a severe complication of blood transfusion that is thought of as a two-hit event: first the underlying patient condition (e.g., sepsis), and then the transfusion. Transfusion factors include human leukocyte antigen antibodies or biologic response modifiers (BRMs) accumulating during storage. Preclinical studies show an increased TRALI risk with longer stored platelets, clinical studies are conflicting. We aim to discover whether longer platelet concentrate (PC) storage time increases TRALI risk in a controlled human experiment. STUDY DESIGN AND METHODS: In a randomized controlled trial, 18 healthy male volunteers received a first hit of experimental endotoxemia (2 ng/kg lipopolysaccharide), and a second hit of fresh (2-day old) or aged (7-day old) autologous PC, or physiological saline. After 6 h, changes in TRALI pathways were determined using spirometry, chest X-ray, and bronchoalveolar lavage (BAL). RESULTS: All subjects reacted adequately to lipopolysaccharide infusion and satisfied SIRS criteria (increased pulse [>90/min] and temperature [>38°C]). There were no differences between the saline, fresh, and aged PC groups in BAL-fluid protein (95 ± 33 µg/ml; 83 ± 21 µg/ml and 104 ± 29 µg/ml, respectively) and relative neutrophil count (1.5 ± 0.5%; 1.9 ± 0.8% and 1.3 ± 0.8%, respectively), nor in inflammatory BAL-fluid BRMs (Interleukin-6, CXCL8, TNFα , and myeloperoxidase), clinical respiratory parameters, and spirometry results. All chest X-rays were normal. CONCLUSIONS: In a human endotoxemia model of autologous platelet transfusion, with an adequate first hit and platelet storage lesion, transfusion of 7-day-old PC does not increase pulmonary inflammation compared with 2-day-old PC.

Systemic inhibition of the membrane attack complex impedes neuroinflammation in chronic relapsing experimental autoimmune encephalomyelitis.

The complement system is a key driver of neuroinflammation. Activation of complement by all pathways, results in the formation of the anaphylatoxin C5a and the membrane attack complex (MAC). Both initiate pro-inflammatory responses which can contribute to neurological disease. In this study, we delineate the specific roles of C5a receptor signaling and MAC formation during the progression of experimental autoimmune encephalomyelitis (EAE)-mediated neuroinflammation. MAC inhibition was achieved by subcutaneous administration of an antisense oligonucleotide specifically targeting murine C6 mRNA (5 mg/kg). The C5a receptor 1 (C5aR1) was inhibited with the C5a receptor antagonist PMX205 (1.5 mg/kg). Both treatments were administered systemically and started after disease onset, at the symptomatic phase when lymphocytes are activated. We found that antisense-mediated knockdown of C6 expression outside the central nervous system prevented relapse of disease by impeding the activation of parenchymal neuroinflammatory responses, including the Nod-like receptor protein 3 (NLRP3) inflammasome. Furthermore, C6 antisense-mediated MAC inhibition protected from relapse-induced axonal and synaptic damage. In contrast, inhibition of C5aR1-mediated inflammation diminished expression of major pro-inflammatory mediators, but unlike C6 inhibition, it did not stop progression of neurological disability completely. Our study suggests that MAC is a key driver of neuroinflammation in this model, thereby MAC inhibition might be a relevant treatment for chronic neuroinflammatory diseases.

LRSAM1-mediated ubiquitylation is disrupted in axonal Charcot-Marie-Tooth disease 2P.

Charcot-Marie-Tooth (CMT) disease type 2 is a genetically heterogeneous group of inherited neuropathies characterized by motor and sensory deficits as a result of peripheral axonal degeneration. We recently reported a frameshift (FS) mutation in the Really Interesting New Gene finger (RING) domain of LRSAM1 (c.2121_2122dup, p.Leu708Argfs) that encodes an E3 ubiquitin ligase, as the cause of axonal-type CMT (CMT2P). However, the frequency of LRSAM1 mutations in CMT2 and the functional basis for their association with disease remains unknown. In this study, we evaluated LRSAM1 mutations in two large Dutch cohorts. In the first cohort (n = 107), we sequenced the full LRSAM1 coding exons in an unbiased fashion, and, in the second cohort (n = 468), we specifically sequenced the last, RING-encoding exon in individuals where other CMT-associated genes had been ruled out. We identified a novel LRSAM1 missense mutation (c.2120C > T, p.Pro707Leu) mapping to the RING domain. Based on our genetic analysis, the occurrence of pathogenic LRSAM1 mutations is estimated to be rare. Functional characterization of the FS, the identified missense mutation, as well as of another recently reported pathogenic missense mutation (c.2081G > A, p.Cys694Tyr), revealed that in vitro ubiquitylation activity was largely abrogated. We demonstrate that loss of the E2-E3 interaction that is an essential prerequisite for supporting ubiquitylation of target substrates, underlies this reduced ubiquitylation capacity. In contrast, LRSAM1 dimerization and interaction with the bona fide target TSG101 were not disrupted. In conclusion, our study provides further support for the role of LRSAM1 in CMT and identifies LRSAM1-mediated ubiquitylation as a common determinant of disease-associated LRSAM1 mutations.

EXOSC3 mutations in pontocerebellar hypoplasia type 1: novel mutations and genotype-phenotype correlations.

BACKGROUND: Pontocerebellar hypoplasia (PCH) represents a group of neurodegenerative disorders with prenatal onset. Eight subtypes have been described thus far (PCH1-8) based on clinical and genetic features. Common characteristics include hypoplasia and atrophy of the cerebellum, variable pontine atrophy, and severe mental and motor impairments. PCH1 is distinctly characterized by the combination with degeneration of spinal motor neurons. Recently, mutations in the exosome component 3 gene (EXOSC3) have been identified in approximately half of the patients with PCH subtype 1. METHODS: We selected a cohort of 99 PCH patients (90 families) tested negative for mutations in the TSEN genes, RARS2, VRK1 and CASK. Patients in this cohort were referred with a tentative diagnose PCH type 1, 2, 4, 7 or unclassified PCH. Genetic analysis of the EXOSC3 gene was performed using Sanger sequencing. Clinical data, MR images and autopsy reports of patients positive for EXOSC3 mutations were analyzed. RESULTS: EXOSC3 mutations were found in twelve families with PCH subtype 1, and were not found in patients with other PCH subtypes. Identified mutations included a large deletion, nonsense and missense mutations. Examination of clinical data reveals a prolonged disease course in patients with a homozygous p.D132A mutation. MRI shows variable pontine hypoplasia in EXOSC3 mediated PCH, where the pons is largely preserved in patients with a homozygous p.D132A mutation, but attenuated in patients with other mutations. Additionally, bilateral cerebellar cysts were found in patients compound heterozygous for a p.D132A mutation and a nonsense allele. CONCLUSIONS: EXOSC3 mediated PCH shows clear genotype-phenotype correlations. A homozygous p.D132A mutation leads to PCH with possible survival into early puberty, and preservation of the pons. Compound heterozygosity for a p.D132A mutation and a nonsense or p.Y109N allele, a homozygous p.G31A mutation or a p.G135E mutation causes a more rapidly progressive course leading to death in infancy and attenuation of the ventral pons.Our findings imply a clear correlation between genetic mutation and clinical outcome in EXOSC3 mediated PCH, including variable involvement of the pons.

A frameshift mutation in LRSAM1 is responsible for a dominant hereditary polyneuropathy.

Despite the high number of genes identified in hereditary polyneuropathies/Charcot-Marie-Tooth (CMT) disease, the genetic defect in many families is still unknown. Here we report the identification of a new gene for autosomal dominant axonal neuropathy in a large three-generation family. Linkage analysis identified a 5 Mb region on 9q33-34 with a LOD score of 5.12. Sequence capture and next-generation sequencing of the region of interest identified five previously unreported non-synonymous heterozygous single nucleotide changes or indels, four of which were confirmed by Sanger sequencing. Two sequence variants co-segregated with the disease, and one, a 2 bp insertion in the last exon of LRSAM1, was also absent in 676 ethnicity-matched control chromosomes. This frameshift mutation (p.Leu708Argfx28) is located in the C-terminal RING finger motif of the encoded protein. Ubiquitin ligase activity in transfected cells with constructs carrying the patient mutation was affected as measured by a higher level of abundance of TSG101, the only reported target of LRSAM1. Injections of morpholino oligonucleotides in zebrafish embryos directed against the ATG or last splice site of zebrafish Lrsam1 disturbed neurodevelopment, showing a less organized neural structure and, in addition, affected tail formation and movement. LRSAM1 is highly expressed in adult spinal cord motoneurons as well as in fetal spinal cord and muscle tissue. Recently, a homozygous mutation in LRSAM1 was proposed as a strong candidate for the disease in a family with recessive axonal polyneuropathy. Our data strongly support the hypothesis that LRSAM1 mutations can cause both dominant and recessive forms of CMT.

TSEN54 mutations cause pontocerebellar hypoplasia type 5.

Pontocerebellar hypoplasia (PCH) is a group of autosomal recessive neurodegenerative disorders characterized by prenatal onset of stunted brain growth and progressive atrophy predominantly affecting cerebellum, pons and olivary nuclei, and to a lesser extent also the cerebral cortex. Six subtypes (PCH1-6) were described and genes for four types (PCH1, 2, 4 and 6) were identified. Mutations in the tRNA splicing endonuclease subunit (TSEN) genes 54, 2 and 34 are found in PCH2 and PCH4. One family with severe prenatal onset of PCH has been the only representative of PCH5 published so far, and the molecular genetic status of PCH5 has not been ascertained until now. We screened the previously reported PCH5 family for mutations in the TSEN54 gene. The PCH5 patient was found to be the result of compound heterozygosity for the common TSEN54 mutation (p.A307S) plus a novel splice site mutation. The mutations associated with PCH5 are similar to what has been reported in PCH4. Thus, PCH5, PCH4 and PCH2 represent a spectrum of clinical manifestations caused by different mutations in the TSEN genes. We, therefore, propose to classify PCH2, PCH4 and PCH5 as TSEN mutation spectrum disorders.

Evaluation of locked nucleic acid-modified small interfering RNA in vitro and in vivo.

RNA interference has become widely used as an experimental tool to study gene function. In addition, small interfering RNA (siRNA) may have great potential for the treatment of diseases. Recently, it was shown that siRNA can be used to mediate gene silencing in mouse models. Locally administered siRNAs entered the first clinical trials, but strategies for successful systemic delivery of siRNA are still under development. Challenges still exist about the stability, delivery, and therapeutic efficacy of siRNA. In the present study, we compare the efficacy of two methods of systemic siRNA delivery and the effects of siRNA modifications using locked nucleic acids (LNA) in a xenograft cancer model. Low volume tail vein bolus injections and continuous s.c. delivery using osmotic minipumps yielded similar uptake levels of unmodified siRNA by tumor xenografts. Both routes of administration mediated sequence-specific inhibition of two unrelated targets inside tumor xenografts. Previous studies have shown that LNA can be incorporated into the sense strand of siRNA while the efficacy is retained. Modification of siRNA targeting green fluorescent protein with LNA results in a significant increase in serum stability and thus may be beneficial for clinical applications. We show that minimal 3' end LNA modifications of siRNA are effective in stabilization of siRNA. Multiple LNA modifications in the accompanying strand further increase the stability but negate the efficacy in vitro and in vivo. In vivo, LNA-modified siRNA reduced off-target gene regulation compared with nonmodified siRNA. End-modified siRNA targeting green fluorescent protein provides a good trade-off between stability and efficacy in vivo using the two methods of systemic delivery in the nude mouse model. Therefore, LNA-modified siRNA should be preferred over unmodified siRNA.

On the in vitro and in vivo properties of four locked nucleic acid nucleotides incorporated into an anti-H-Ras antisense oligonucleotide.

Locked nucleic acid (beta-D-LNA) monomers are conformationally restricted nucleotides bearing a methylene 2'-O, 4'-C linkage that have an unprecedented high affinity for matching DNA or RNA. In this study, we compared the in vitro and in vivo properties of four different LNAs, beta-D-amino LNA (amino-LNA), beta-D-thio LNA (thio-LNA), beta-D-LNA (LNA), and its stereoisomer alpha-L-LNA in an antisense oligonucleotide (ODN). A well-known antisense ODN design against H-Ras was modified at the 5'- and 3'-ends with the different LNA analogues (LNA-DNA-LNA gapmer design). The resulting gapmers were tested in cancer-cell cultures and in a nude-mouse model bearing prostate tumor xenografts. The efficacy in target knockdown, the biodistribution, and the ability to inhibit tumor growth were measured. All anti H-Ras ODNs were very efficient in H-Ras mRNA knockdown in vitro, reaching maximum effect at concentrations below 5 nM. Moreover, the anti-H-Ras ODN containing alpha-L-LNA had clearly the highest efficacy in H-Ras knockdown. All LNA types displayed a great stability in serum. ODNs containing amino-LNA showed an increased uptake by heart, liver, and lungs as compared to the other LNA types. Both alpha-L-LNA and LNA gapmer ODNs had a high efficacy of tumor-growth inhibition and were nontoxic at the tested dosages. Remarkably, in vivo tumor-growth inhibition could be observed at dosages as low as 0.5 mg kg(-1) per day. These results indicate that alpha-L-LNA is a very promising member of the family of LNA analogues in antisense applications.

In vivo tumor growth inhibition and biodistribution studies of locked nucleic acid (LNA) antisense oligonucleotides.

Locked nucleic acids (LNA) are novel high-affinity DNA analogs that can be used as genotype-specific drugs. The LNA oligonucleotides (LNA PO ODNs) are very stable in vitro and in vivo without the need for a phosphorothiolated backbone. In this study we tested the biological fate and the efficacy in tumor growth inhibition of antisense oligonucleotides directed against the gene of the large subunit of RNA polymerase II (POLR2A) that are completely synthesized as LNA containing diester backbones. These full LNA oligonucleotides strongly reduce POLR2A protein levels. Full LNA PO ODNs appeared to be very stable compounds when injected into the circulation of mice. Full LNA PO ODNs were continuously administered for 14 days to tumor-bearing nude mice. Tumor growth was inhibited sequence specifically at dosages from 1 mg/kg/day. LNA PO ODNs appeared to be non-toxic at dosages <5 mg/kg/day. Biodistribution studies showed the kidneys to have the highest uptake of LNA PO ODNs and urinary secretion as the major route of clearance. This report shows that LNA PO ODNs are potent genotype-specific drugs that can inhibit tumor growth in vivo.