An AP4B1 frameshift mutation in siblings with intellectual disability and spastic tetraplegia further delineates the AP-4 deficiency syndrome

The recently proposed adaptor protein 4 (AP-4) deficiency syndrome comprises a group of congenital neurological disorders characterized by severe intellectual disability (ID), delayed or absent speech, hereditary spastic paraplegia, and growth retardation. AP-4 is a heterotetrameric protein complex with important functions in vesicle trafficking. Mutations in genes affecting different subunits of AP-4, including AP4B1, AP4E1, AP4S1, and AP4M1, have been reported in patients with the AP-4 deficiency phenotype. We describe two siblings from a non-consanguineous couple who presented with severe ID, absent speech, microcephaly, growth retardation, and progressive spastic tetraplegia. Whole-exome sequencing in the two patients identified the novel homozygous 2-bp deletion c.1160_1161delCA (p.(Thr387Argfs*30)) in AP4B1. Sanger sequencing confirmed the mutation in the siblings and revealed it in the heterozygous state in both parents. The AP4B1-associated phenotype has previously been assigned to spastic paraplegia-47. Identification of a novel AP4B1 alteration in two patients with clinical manifestations highly similar to other individuals with mutations affecting one of the four AP-4 subunits further supports the observation that loss of AP-4 assembly or functionality underlies the common clinical features in these patients and underscores the existence of the clinically recognizable AP-4 deficiency syndrome.     

The effects of endothelial injury on smooth muscle cell proliferation

The endothelial injury induced by the placement of a synthetic graft has been implicated as a stimulus for the development of MIH. In this study we compared the degree of EC coverage and the early SMC-PR in the arterial segments proximal and distal to 2 mm diameter PTFE grafts that had been placed in rabbit carotid arteries (n = 49). In vivo labeling with 3H-thymidine and Evans blue was carried out at intervals of 2 to 33 days after grafting. The SMC-PR was measured as the degree of 3H-labeled DNA divided by the total DNA for each segment, and the EC coverage was determined by planimetry of the area of Evans blue exclusion. There was an early rise in the SMC-PR in both arterial segments, but it was more marked in the distal segment (p less than 0.001). There was no correlation between the SMC-PR and the degree of EC coverage in either the proximal (r = 0.25) or the distal segments (r = 0.10). The data suggest that there is a greater SMC-PR at the distal end of an implanted PTFE graft. The degree of endothelial loss and its regrowth does not appear to be an important factor.     

Lateral view of the thorax: the profile

The lateral view of the chest is a complementary incidence performed less frequently nowadays with the great frequency of chest CT. In fact, this lateral radiograph has important potential and can even give some exclusive information. With the 3-dimensional visualization provided by CT, the lateral radiograph of the chest is even easier to understand. Following in the footsteps of our great predecessors (Felson, Heitzman, Proto) we propose the left lateral view and offer our opinion about indications, techniques and results. The left lateral view can be performed with perfect perpendicular orientation or with a slight lateral shift that can be chosen with the right shoulder forwards (shifted right anterior lateral) or the contrary (shifted left anterior lateral). The left lateral view, like the antero-posterior view must be interpreted in a circular-concentric fashion.     

Toll-like receptor 9 signaling mediates the anti-inflammatory effects of probiotics in murine experimental colitis

BACKGROUND & AIMS: We tested whether the attenuation of experimental colitis by live probiotic bacteria is due to their immunostimulatory DNA, whether toll-like receptor (TLR) signaling is required, and whether nonviable probiotics are effective. METHODS: Methylated and unmethylated genomic DNA isolated from probiotics (VSL-3), DNAse-treated probiotics and Escherichia coli (DH5 alpha) genomic DNA were administered intragastrically (i.g.) or subcutaneously (s.c.) to mice prior to the induction of colitis. Viable or gamma-irradiated probiotics were administered i.g. to wild-type mice and mice deficient in different TLR or in the adaptor protein MyD88, 10 days prior to administration of dextran sodium sulfate (DSS) to their drinking water and for 7 days thereafter. RESULTS: Intragastric and s.c. administration of probiotic and E. coli DNA ameliorated the severity of DSS-induced colitis, whereas methylated probiotic DNA, calf thymus DNA, and DNase-treated probiotics had no effect. The colitis severity was attenuated to the same extent by i.g. delivery of nonviable gamma-irradiated or viable probiotics. Mice deficient in MyD88 did not respond to gamma-irradiated probiotics. The severity of DSS-induced colitis in TLR2 and TLR4 deficient mice was significantly decreased by i.g. administration of gamma-irradiated probiotics, whereas, in TLR9-deficient mice, gamma-irradiated probiotics had no effect. CONCLUSIONS: The protective effects of probiotics are mediated by their own DNA rather than by their metabolites or ability to colonize the colon. TLR9 signaling is essential in mediating the anti-inflammatory effect of probiotics, and live microorganisms are not required to attenuate experimental colitis because nonviable probiotics are equally effective.     

Improving the Quality of EDTA-treated Blood Specimens from Mice

Nonterminal blood sampling in laboratory mice is a very common procedure. With the goal of improving animal welfare, different sampling sites and methods have been compared but have not achieved a consensus. Moreover, most of these studies overlooked the quality of blood specimens collected. The main preanalytical concern with EDTA-treated blood specimens for hematology analyses is platelet aggregation, which is known to cause analytical errors. Our objective was to find a nonterminal blood sampling method with minimal adverse effects on mice and few or no platelet aggregates. We tested and compared 2 collection sites, 4 sampling methods, and 3 antithrombotic drugs in 80 C57BL6/j male and female mice by evaluating platelet aggregates on blood smears and platelet, WBC, and RBC counts. In addition, the blood collection process was carefully evaluated, and adverse effects were recorded. Platelet aggregation was lower in specimens collected from the jugular vein than from the facial vein, with no effect of the sampling device or the presence of an antithrombotic additive. Highly aggregated specimens were significantly associated with lower platelet counts, whereas aggregation had no effect on WBC or RBC counts. Adverse events during sampling were significantly associated with more numerous platelet aggregates. The jugular vein is thus a satisfactory sampling site in mice in terms of both animal welfare and low platelet aggregation. Using antithrombotic agents appears to be unnecessary, whereas improving sampling conditions remains a key requirement to ensure the quality of EDTA-treated blood specimens from mice.

Industrial and academic research often require hematology analyses of mouse blood. Consequently, many terminal and nonterminal techniques have become available for blood sampling in mice.^{12,21,27,40,42,53} Preanalytical variation in clinical pathology is known to be a major issue.^5,45,49 Although the effects of the blood sampling method on animal welfare have been the subject of many preanalytical hematology and biochemical analyses,^{1,6,8,9,15,16,18,24-26,36,47,50-52,54} no agreement has been reached regarding the optimal method for nonterminal blood collection in mice and, to our knowledge, only a few investigations^1,8,15,16,18 have addressed the quality of the resulting blood specimens.Our own experience of hematology measurements from nonterminal mouse EDTA-blood specimens is that some specimens show both visible clots and platelet aggregation, the latter being detected only from microscopic examination of blood smears.³³ Whereas specimens with visible clots can be eliminated, microscopic platelet aggregates can also interfere with hematology analyses or cause analytical errors, as has been reported in other species including cats.^13,22,31,39 These abnormalities require repeat sampling when possible; otherwise, the number of validated results is decreased. EDTA-treated mouse blood is especially prone to platelet aggregation and clotting.^14,28,43 This characteristic leads to errors in platelet counts (pseudothrombocytopenia) and possible misidentification of platelet aggregates as eosinophils, resulting in false leukocytosis and eosinophilia.¹⁴ In vitro platelet aggregation in mice is due to high platelet counts^34,43 and is influenced by numerous preanalytical factors including the sampling method, collection site, specimen processing, anticoagulant used, the blood:anticoagulant ratio, the mouse strain and genetic alterations.^19,28,30,43 The literature on the influence of preanalytical factors on the quality of CBC analyses in mice is scant,⁴³ and no agreement has yet been reached regarding the optimal method for nonterminal blood collection in mice. In humans and various animal species, platelet aggregation can be reduced by adding platelet aggregation inhibitors that act at different steps of aggregation. To our knowledge, the addition of such inhibitors to mouse whole blood has not been tested as a means to improve the quality of mice EDTA-treated blood specimens.The aim of this study was therefore to identify the best preanalytical conditions for nonterminal blood collection in mice, based on animal welfare, scores of platelet aggregation, and platelet, RBC, and WBC counts. The hypotheses we tested were that 1) adding an antithrombotic drug (or multiple such drugs) to the EDTA-treated blood specimen would prevent or at least significantly lower platelet aggregation, 2) the site and the method of collection influence in vitro platelet aggregation, and 3) high-quality blood sampling is a key to reducing platelet aggregation in blood specimens.