Consequences of excessive glucosylsphingosine in glucocerebrosidase-deficient zebrafish.

In Gaucher disease (GD), the deficiency of glucocerebrosidase causes lysosomal accumulation of glucosylceramide (GlcCer), which is partly converted by acid ceramidase to glucosylsphingosine (GlcSph) in the lysosome. Chronically elevated blood and tissue GlcSph is thought to contribute to symptoms in GD patients as well as to increased risk for Parkinson’s disease. On the other hand, formation of GlcSph may be beneficial since the water soluble sphingoid base is excreted via urine and bile. To study the role of excessive GlcSph formation during glucocerebrosidase deficiency, we studied zebrafish that have two orthologs of acid ceramidase, Asah1a and Asah1b. Only the latter is involved in the formation of GlcSph in glucocerebrosidase-deficient zebrafish as revealed by knockouts of Asah1a or Asah1b with glucocerebrosidase deficiency (either pharmacologically induced or genetic). Comparison of zebrafish with excessive GlcSph (gba1^-/- fish) and without GlcSph (gba1^-/-:asah1b^-/- fish) allowed us to study the consequences of chronic high levels of GlcSph. Prevention of excessive GlcSph in gba1^-/-:asah1b^-/- fish did not restrict storage cells, GlcCer accumulation, or neuroinflammation. However, GD fish lacking excessive GlcSph show an ameliorated course of disease reflected by significantly increased lifespan, delayed locomotor abnormality, and delayed development of an abnormal curved back posture. The loss of tyrosine hydroxylase 1 (th1) mRNA, a marker of dopaminergic neurons, is slowed down in brain of GD fish lacking excessive GlcSph. In conclusion, in the zebrafish GD model, excess GlcSph has little impact on (neuro)inflammation or the presence of GlcCer-laden macrophages but rather seems harmful to th1-positive dopaminergic neurons.     

Msx2 is an immediate downstream effector of Pax3 in the development of the murine cardiac neural crest.

The neural crest plays a crucial part in cardiac development. Cells of the cardiac subpopulation of cranial neural crest migrate from the hindbrain into the outflow tract of the heart where they contribute to the septum that divides the pulmonary and aortic channels. In Splotch mutant mice, which lack a functional Pax3 gene, migration of cardiac neural crest is deficient and aorticopulmonary septation does not occur. Downstream genes through which Pax3 regulates cardiac neural crest development are unknown. Here, using a combination of genetic and molecular approaches, we show that the deficiency of cardiac neural crest development in the Splotch mutant is caused by upregulation of Msx2, a homeobox gene with a well-documented role as a regulator of BMP signaling. We provide evidence, moreover, that Pax3 represses Msx2 expression via a direct effect on a conserved Pax3 binding site in the Msx2 promoter. These results establish Msx2 as an effector of Pax3 in cardiac neural crest development.     

Spinal Radiographic Progression in Patients with Ankylosing Spondylitis Treated with TNF-α Blocking Therapy: A Prospective Longitudinal Observational Cohort Study

Objectives

To evaluate spinal radiographic damage over time and to explore the associations of radiographic progression with patient characteristics and clinical assessments including disease activity in ankylosing spondylitis (AS) patients treated with tumor necrosis factor-alpha (TNF-α) blocking therapy in daily clinical practice.

Methods

Consecutive outpatients from the Groningen Leeuwarden AS (GLAS) cohort were included based on the availability of cervical and lumbar radiographs before start of TNF-α blocking therapy and after 2, 4, and/or 6 years of follow-up. Clinical data were assessed at the same time points. Radiographs were scored by two independent readers using the modified Stoke AS Spine Score (mSASSS). Spinal radiographic progression in relation to clinical assessments was analyzed using generalized estimating equations.

Results

176 AS patients were included, 58% had syndesmophytes at baseline. Median mSASSS increased significantly from 10.7 (IQR: 4.6–24.0) at baseline to 14.8 (IQR: 7.9–32.8) at 6 years. At the group level, spinal radiographic progression was linear with a mean progression rate of 1.3 mSASSS units per 2 years. Both spinal radiographic damage at baseline and radiographic progression were highly variable between AS patients. Male gender, older age, longer disease duration, higher BMI, longer smoking duration, high CRP, and high ASDAS were significantly associated with syndesmophytes at baseline. Significantly more radiographic progression was seen in patients with versus without syndesmophytes (2.0 vs. 0.5 mSASSS units per 2 years) and in patients >40 versus ≤40 years of age (1.8 vs. 0.7 mSASSS units per 2 years). No longitudinal associations between radiographic progression and clinical assessments were found.

Conclusions

This prospective longitudinal observational cohort study in daily clinical practice shows overall slow and linear spinal radiographic progression in AS patients treated with TNF-α blocking therapy. At the individual level, progression was highly variable. Patients with syndesmophytes at baseline showed a 4-fold higher radiographic progression rate than patients without syndesmophytes.     

Temporal brow lift using botulinum toxin A

The objective of this study was to determine whether brow elevation occurs as a result of paralysis of brow depressors after botulinum toxin A injection. The study's design was a prospective case series with pretreatment and posttreatment outcome evaluation with statistical analysis at a university-based division of facial plastic surgery private clinic. Twenty-two patients of a consecutive sample desiring a cosmetic enhancement underwent injection of botulinum toxin A directed to brow depressors. Injections consisted of 7 to 10 units of botulinum toxin A (Botox, Allergan, Irvine, Calif.) into selected brow depressor muscle (lateral orbicularis oculi) bilaterally. No patients withdrew for adverse effects. All patients were evaluated 2 weeks after treatment. The outcomes were measured by change in brow elevation along vertical axis extending from both midpupil and lateral canthus to the caudal row of brow hairs with eyes at neutral gaze and the head at Frankfort plane. Preintervention and postintervention brow height was measured by the primary clinical investigator. The average brow elevation from the midpupil observed after selected injection of brow depressors with botulinum toxin A was 1.02 mm (p = 0.038). The average brow elevation from the lateral canthus observed after selected injection of brow depressors with botulinum toxin A was 4.83 mm (p<0.0001). Significant temporal brow elevation occurs as the result of paralysis of brow depressors by using botulinum toxin A injection. This procedure may be considered an alternative to surgical brow elevation.     

Guillain-Barré Syndrome and Adjuvanted Pandemic Influenza A (H1N1) 2009 Vaccines: A Multinational Self-Controlled Case Series in Europe

Background

The risk of Guillain-Barré syndrome (GBS) following the United States'' 1976 swine flu vaccination campaign in the USA led to enhanced active surveillance during the pandemic influenza (A(H1N1)pdm09) immunization campaign. This study aimed to estimate the risk of GBS following influenza A(H1N1)pdm09 vaccination.

Methods

A self-controlled case series (SCCS) analysis was performed in Denmark, Finland, France, Netherlands, Norway, Sweden, and the United Kingdom. Information was collected according to a common protocol and standardised procedures. Cases classified at levels 1–4a of the Brighton Collaboration case definition were included. The risk window was 42 days starting the day after vaccination. Conditional Poisson regression and pooled random effects models estimated adjusted relative incidences (RI). Pseudo likelihood and vaccinated-only methods addressed the potential contraindication for vaccination following GBS.

Results

Three hundred and three (303) GBS and Miller Fisher syndrome cases were included. Ninety-nine (99) were exposed to A(H1N1)pdm09 vaccination, which was most frequently adjuvanted (Pandemrix and Focetria). The unadjusted pooled RI for A(H1N1)pdm09 vaccination and GBS was 3.5 (95% Confidence Interval (CI): 2.2–5.5), based on all countries. This lowered to 2.0 (95% CI: 1.2–3.1) after adjustment for calendartime and to 1.9 (95% CI: 1.1–3.2) when we accounted for contra-indications. In a subset (Netherlands, Norway, and United Kingdom) we further adjusted for other confounders and there the RI decreased from 1.7 (adjusted for calendar month) to 1.4 (95% CI: 0.7–2.8), which is the main finding.

Conclusion

This study illustrates the potential of conducting European collaborative vaccine safety studies. The main, fully adjusted analysis, showed that the RI of GBS was not significantly elevated after influenza A(H1N1)pdm09 vaccination (RI = 1.4 (95% CI: 0.7–2.8). Based on the upper limits of the pooled estimate we can rule out with 95% certainty that the number of excess GBS cases after influenza A(H1N1)pdm09 vaccination would be more than 3 per million vaccinated.     

Gibberellins in Leaves of Alstroemeria hybrida: Identification and Quantification in Relation to Leaf Age

In alstroemeria (Alstroemeria hybrida), leaf senescence is retarded effectively by the application of gibberellins (GAs). To study the role of endogenous GAs in leaf senescence, the GA content was analyzed by combined gas chromatography and mass spectrometry. Five 13-hydroxy GAs (GA₁₉, GA₂₀, GA₁, GA₈, and GA₂₉) and three non-13-hydroxy GAs (GA₉ and GA₄) were identified in leaf extracts by comparing Kováts retention indices (KRIs) and full scan mass sprectra with those of reference GAs. In addition, GA₁₅, GA₄₄, GA₂₄, and GA₃₄ were tentatively identified by comparing selected ion monitoring results and KRIs with those of reference GAs. A number of GAs were detected in conjugated form as well. Concentrations of GAs in alstroemeria changed with the development of leaves. The proportion of biologically active GA₁ and GA₄ decreased with progressive senescence and the fraction of conjugated GAs increased. Received May 26, 1997; accepted August 12, 1997