Insights into post-translational processing of beta-galactosidase in an animal model resembling late infantile human G-gangliosidosis

G(M1)-gangliosidosis is a lysosomal storage disorder caused by a deficiency of ss-galactosidase activity. Human GM1-gangliosidosis has been classified into three forms according to the age of clinical onset and specific biochemical parameters. In the present study, a canine model for type II late infantile human GM1-gangliosidosis was investigated 'in vitro' in detail. For a better understanding of the molecular pathogenesis underlying G(M1)-gangliosidosis the study focused on the analysis of the molecular events and subsequent intracellular protein trafficking of beta-galactosidase. In the canine model the genetic defect results in exclusion or inclusion of exon 15 in the mRNA transcripts and to translation of two mutant precursor proteins. Intracellular localization, processing and enzymatic activity of these mutant proteins were investigated. The obtained results suggested that the beta-galactosidase C-terminus encoded by exons 15 and 16 is necessary for correct C-terminal proteolytic processing and enzyme activity but does not affect the correct routing to the lysosomes. Both mutant protein precursors are enzymatically inactive, but are transported to the lysosomes clearly indicating that the amino acid sequences encoded by exons 15 and 16 are necessary for correct folding and association with protective protein/cathepsin A, whereas the routing to the lysosomes is not influenced. Thus, the investigated canine model is an appropriate animal model for the human late infantile form and represents a versatile system to test gene therapeutic approaches for human and canine G(M1)-gangliosidosis.

A duplication in the canine beta-galactosidase gene GLB1 causes exon skipping and GM1-gangliosidosis in Alaskan huskies

GM(1)-gangliosidosis is a lysosomal storage disease that is inherited as an autosomal recessive disorder, predominantly caused by structural defects in the beta-galactosidase gene (GLB1). The molecular cause of GM(1)-gangliosidosis in Alaskan huskies was investigated and a novel 19-bp duplication in exon 15 of the GLB1 gene was identified. The duplication comprised positions +1688-+1706 of the GLB1 cDNA. It partially disrupted a potential exon splicing enhancer (ESE), leading to exon skipping in a fraction of the transcripts. Thus, the mutation caused the expression of two different mRNAs from the mutant allele. One transcript contained the complete exon 15 with the 19-bp duplication, while the other transcript lacked exon 15. In the transcript containing exon 15 with the 19-bp duplication a premature termination codon (PTC) appeared, but due to its localization in the last exon of canine GLB1, nonsense-mediated RNA decay (NMD) did not occur. As a consequence of these molecular events two different truncated GLB1 proteins are predicted to be expressed from the mutant GLB1 allele. In heterozygous carrier animals the wild-type allele produces sufficient amounts of the active enzyme to prevent clinical signs of disease. In affected homozygous dogs no functional GLB1 is synthesized and G(M1)-gangliosidosis occurs.

Infarction Distribution Pattern in Acute Stroke May Predict the Extent of Leptomeningeal Collaterals.

OBJECTIVE The aim of this study was to evaluate whether the distribution pattern of early ischemic changes in the initial MRI allows a practical method for estimating leptomeningeal collateralization in acute ischemic stroke (AIS). METHODS Seventy-four patients with AIS underwent MRI followed by conventional angiogram and mechanical thrombectomy. Diffusion restriction in Diffusion weighted imaging (DWI) and correlated T2-hyperintensity of the infarct were retrospectively analyzed and subdivided in accordance with Alberta Stroke Program Early CT score (ASPECTS). Patients were angiographically graded in collateralization groups according to the method of Higashida, and dichotomized in 2 groups: 29 subjects with collateralization grade 3 or 4 (well-collateralized group) and 45 subjects with grade 1 or 2 (poorly-collateralized group). Individual ASPECTS areas were compared among the groups. RESULTS Means for overall DWI-ASPECTS were 6.34 vs. 4.51 (well vs. poorly collateralized groups respectively), and for T2-ASPECTS 9.34 vs 8.96. A significant difference between groups was found for DWI-ASPECTS (p<0.001), but not for T2-ASPECTS (p = 0.088). Regarding the individual areas, only insula, M1-M4 and M6 showed significantly fewer infarctions in the well-collateralized group (p-values <0.001 to 0.015). 89% of patients in the well-collateralized group showed 0-2 infarctions in these six areas (44.8% with 0 infarctions), while 59.9% patients of the poor-collateralized group showed 3-6 infarctions. CONCLUSION Patients with poor leptomeningeal collateralization show more infarcts on the initial MRI, particularly in the ASPECTS areas M1 to M4, M6 and insula. Therefore DWI abnormalities in these areas may be a surrogate marker for poor leptomeningeal collaterals and may be useful for estimation of the collateral status in routine clinical evaluation.

Mechanical Thrombectomy for Isolated M2 Occlusions: A Post Hoc Analysis of the STAR, SWIFT, and SWIFT PRIME Studies.

BACKGROUND AND PURPOSE Mechanical thrombectomy is beneficial for patients with acute ischemic stroke and a proximal anterior occlusion, but it is unclear if these results can be extrapolated to patients with an M2 occlusion. The purpose of this study was to examine the technical aspects, safety, and outcomes of mechanical thrombectomy with a stent retriever in patients with an isolated M2 occlusion who were included in 3 large multicenter prospective studies. MATERIALS AND METHODS We included patients from the Solitaire Flow Restoration Thrombectomy for Acute Revascularization (STAR), Solitaire With the Intention For Thrombectomy (SWIFT), and Solitaire With the Intention for Thrombectomy as Primary Endovascular Treatment (SWIFT PRIME) studies, 3 large multicenter prospective studies on thrombectomy for ischemic stroke. We compared outcomes and technical details of patients with an M2 with those with an M1 occlusion. All patients were treated with a stent retriever. Imaging data and outcomes were scored by an independent core laboratory. Successful reperfusion was defined as modified Thrombolysis in Cerebral Infarction score of 2b/3. RESULTS We included 50 patients with an M2 and 249 patients with an M1 occlusion. Patients with an M2 occlusion were older (mean age, 71 versus 67 years; P = .04) and had a lower NIHSS score (median, 13 versus 17; P < .001) compared with those with an M1 occlusion. Procedural time was nonsignificantly shorter in patients with an M2 occlusion (median, 29 versus 35 minutes; P = .41). The average number of passes with a stent retriever was also nonsignificantly lower in patients with an M2 occlusion (mean, 1.4 versus 1.7; P = .07). There were no significant differences in successful reperfusion (85% versus 82%, P = .82), symptomatic intracerebral hemorrhages (2% versus 2%, P = 1.0), device-related serious adverse events (6% versus 4%, P = .46), or modified Rankin Scale score 0-2 at follow-up (60% versus 56%, P = .64). CONCLUSIONS Endovascular reperfusion therapy appears to be feasible in selected patients with ischemic stroke and an M2 occlusion.

Occlusion Location of Middle Cerebral Artery Stroke and Outcome after Endovascular Treatment.

BACKGROUND The aim of this study was to analyze the influence of the location of middle cerebral artery (MCA) occlusion on recanalization, complications and outcome after endovascular therapy. METHODS Four-hundred sixty-four patients with acute MCA occlusions were treated with endovascular therapy. RESULTS Two-hundred ninety-three patients had M1 occlusions, 116 had M2, and 55 had M3/4 occlusions. Partial or complete recanalization was more frequently achieved in M1 (76.8%) than in M2 (59.1%) or M3/4 (47.3%, p < 0.001) occlusions, but favorable outcome (modified Rankin Scale 0-2) was less frequent in M1 (50.9%) than M2 (63.7%) or M3/4 (72.7%, p = 0.018) occlusions. Symptomatic intracerebral hemorrhage (ICH) did not differ between occlusion sites, but asymptomatic ICH was more common in M1 (22.6%) than in M2 occlusions (8.6%, p = 0.003). Recanalization was associated with favorable outcome in M1 (p < 0.001) and proximal M2 (p = 0.003) but not in distal M2 or M3/4 occlusions. CONCLUSIONS Recanalization with endovascular therapy was more frequently achieved in patients with proximal than distal MCA occlusions, but recanalization was associated with favorable outcome only in M1 and proximal M2 occlusions. Outcome was better with distal than proximal occlusions. This study shows that recanalization can be used as a surrogate marker for clinical outcome only in patients with proximal occlusions.

Effect of endovascular reperfusion in relation to site of arterial occlusion

OBJECTIVE To assess whether the association between reperfusion and improved clinical outcomes after stroke differs depending on the site of the arterial occlusive lesion (AOL). METHODS We pooled data from Solitaire With the Intention for Thrombectomy (SWIFT), Solitaire FR Thrombectomy for Acute Revascularisation (STAR), Diffusion and Perfusion Imaging Evaluation for Understanding Stroke Evolution Study 2 (DEFUSE 2), and Interventional Management of Stroke Trial (IMS III) to compare the strength of the associations between reperfusion and clinical outcomes in patients with internal carotid artery (ICA), proximal middle cerebral artery (MCA) (M1), and distal MCA (M2/3/4) occlusions. RESULTS Among 710 included patients, the site of the AOL was the ICA in 161, the proximal MCA in 389, and the distal MCA in 160 patients (M2 = 131, M3 = 23, and M4 = 6). Reperfusion was associated with an increase in the rate of good functional outcome (modified Rankin Scale [mRS] score 0-2) in patients with ICA (odds ratio [OR] 3.5, 95% confidence interval [CI] 1.7-7.2) and proximal MCA occlusions (OR 6.2, 95% CI 3.8-10.2), but not in patients with distal MCA occlusions (OR 1.4, 95% CI 0.8-2.6). Among patients with M2 occlusions, a subset of the distal MCA cohort, reperfusion was associated with excellent functional outcome (mRS 0-1; OR 2.2, 95% CI 1.0-4.7). CONCLUSIONS The association between endovascular reperfusion and better clinical outcomes is more profound in patients with ICA and proximal MCA occlusions compared to patients with distal MCA occlusions. Because there are limited data from randomized controlled trials on the effect of endovascular therapy in patients with distal MCA occlusions, these results underscore the need for inclusion of this subgroup in future endovascular therapy trials.

Influence of unilateral maxillary first molar extraction treatment on second and third molar inclination in Class II subdivision patients.

OBJECTIVE To assess the maxillary second molar (M2) and third molar (M3) inclination following orthodontic treatment of Class II subdivision malocclusion with unilateral maxillary first molar (M1) extraction. MATERIALS AND METHODS Panoramic radiographs of 21 Class II subdivision adolescents (eight boys, 13 girls; mean age, 12.8 years; standard deviation, 1.7 years) before treatment, after treatment with extraction of one maxillary first molar and Begg appliances and after at least 1.8 years in retention were retrospectively collected from a private practice. M2 and M3 inclination angles (M2/ITP, M2/IOP, M3/ITP, M3/IOP), constructed by intertuberosity (ITP) and interorbital planes (IOP), were calculated for the extracted and nonextracted segments. Random effects regression analysis was performed to evaluate the effect on the molar angulation of extraction, time, and gender after adjusting for baseline measurements. RESULTS Time and extraction status were significant predictors for M2 angulation. M2/ITP and M2/IOP decreased by 4.04 (95% confidence interval [CI]: -6.93, 1.16; P  =  .001) and 3.67 (95% CI: -6.76, -0.58; P  =  .020) in the extraction group compared to the nonextraction group after adjusting for time and gender. The adjusted analysis showed that extraction was the only predictor for M3 angulation that reached statistical significance. M3 mesial inclination increased by 7.38° (95% CI: -11.2, -3.54; P < .001) and 7.33° (95% CI: -11.48, -3.19; P  =  .001). CONCLUSIONS M2 and M3 uprighting significantly improved in the extraction side after orthodontic treatment with unilateral maxillary M1 extraction. There was a significant increase in mesial tipping of maxillary second molar crowns over time.

Long-term evaluation of Class II subdivision treatment with unilateral maxillary first molar extraction

OBJECTIVE To evaluate the long-term effects of asymmetrical maxillary first molar (M1) extraction in Class II subdivision treatment. MATERIALS AND METHODS Records of 20 Class II subdivision whites (7 boys, 13 girls; mean age, 13.0 years; SD, 1.7 years) consecutively treated with the Begg technique and M1 extraction, and 15 untreated asymmetrical Class II adolescents (4 boys, 11 girls; mean age, 12.2 years; SD, 1.3 years) were examined in this study. Cephalometric analysis and PAR assessment were carried out before treatment (T1), after treatment (T2), and on average 2.5 years posttreatment (T3) for the treatment group, and at similar time points and average follow-up of 1.8 years for the controls. RESULTS The adjusted analysis indicated that the maxillary incisors were 2.3 mm more retracted in relation to A-Pog between T1 and T3 (β  =  2.31; 95% CI; 0.76, 3.87), whereas the mandibular incisors were 1.3 mm more protracted (β  =  1.34; 95% CI; 0.09, 2.59), and 5.9° more proclined to the mandibular plane (β  =  5.92; 95% CI; 1.43, 10.41) compared with controls. The lower lip appeared 1.4 mm more protrusive relative to the subnasale-soft tissue-Pog line throughout the observation period in the treated adolescents (β  =  1.43; 95% CI; 0.18, 2.67). There was a significant PAR score reduction over the entire follow-up period in the molar extraction group (β  =  -6.73; 95% CI; -10.7, -2.7). At T2, 65% of the subjects had maxillary midlines perfectly aligned with the face. CONCLUSIONS Unilateral M1 extraction in asymmetrical Class II cases may lead to favorable occlusal outcomes in the long term without harming the midline esthetics and soft tissue profile.

OsteoMacs: Key players around bone biomaterials.

Osteal macrophages (OsteoMacs) are a special subtype of macrophage residing in bony tissues. Interesting findings from basic research have pointed to their vast and substantial roles in bone biology by demonstrating their key function in bone formation and remodeling. Despite these essential findings, much less information is available concerning their response to a variety of biomaterials used for bone regeneration with the majority of investigation primarily focused on their role during the foreign body reaction. With respect to biomaterials, it is well known that cells derived from the monocyte/macrophage lineage are one of the first cell types in contact with implanted biomaterials. Here they demonstrate extremely plastic phenotypes with the ability to differentiate towards classical M1 or M2 macrophages, or subsequently fuse into osteoclasts or multinucleated giant cells (MNGCs). These MNGCs have previously been characterized as foreign body giant cells and associated with biomaterial rejection, however more recently their phenotypes have been implicated with wound healing and tissue regeneration by studies demonstrating their expression of key M2 markers around biomaterials. With such contrasting hypotheses, it becomes essential to better understand their roles to improve the development of osteo-compatible and osteo-promotive biomaterials. This review article expresses the necessity to further study OsteoMacs and MNGCs to understand their function in bone biomaterial tissue integration including dental/orthopedic implants and bone grafting materials.