Spinal Muscular Atrophy: position and functional importance of the branch site preceding SMN exon 7

In spinal muscular atrophy, the SMN1 gene is deleted or destroyed by mutation, while the neigbouring, nearly identical SMN2 gene acts as a partial functional substitute. However, due to a single nucleotide exchange, the seventh exon of SMN2 is mostly excluded from the mature mRNA, and the resulting shorter protein is non-functional. Here, we map the previously uncharacterised intron 6 branch point by RT-PCR. Moreover we show that exon 7 inclusion can be either abolished or improved by mutations in this branch site region.

Functional imaging of the parotid glands using blood oxygenation level dependent (BOLD)-MRI at 1.5T and 3T

PURPOSE: To evaluate the function of the parotid glands before and during gustatory stimulation, using an intrinsic susceptibility-weighted MRI method (blood oxygenation level dependent, BOLD-MRI) at 1.5T and 3T. MATERIALS AND METHODS: A total of 10 and 13 volunteers were investigated at 1.5T and 3T, respectively. Measurements were performed before and during gustatory stimulation using ascorbate. Circular regions of interest (ROIs) were delineated in the left and right parotid glands, and in the masseter muscle for comparison. The effects of stimulation were evaluated by calculating the difference between the relaxation rates, DeltaR(2)*. Baseline and stimulation were statistically compared (Student's t-tests), merging both parotid glands. RESULTS: The averaged DeltaR(2)* values prestimulation obtained in all parotid glands were stable (-0.61 to 0.38 x 10(-3) seconds(-1)). At 3T, these values were characterized by an initial drop (to -2.7 x 10(-3) seconds(-1)) followed by a progressive increase toward the baseline. No significant difference was observed between baseline and parotid gland stimulation at 1.5T, neither for the masseter muscle at both field strengths. A considerable interindividual variability (over 76%) was noticed at both magnetic fields. CONCLUSION: BOLD-MRI at 3T was able to detect DeltaR(2)* changes in the parotid glands during gustatory stimulation, consistent with an increase in oxygen consumption during saliva production.

Transplantation of yeast-infected cardiac allografts: a report of 2 cases

For the first time in the literature to date, we report 2 cases of transplantation of yeast-infected cardiac allografts. In both cases, endocardial vegetations were observed before graft implantation. Microbiologic samples grew yeasts: Rhodotorula glutinis was found close to the left atrial appendage in the first case and Candida parapsilosis was identified in a vegetation located at the base of the tricuspid valve in the second case. We discuss the possible routes of donor organ infection and management of these 2 unusual cases.

Outcome and patients' satisfaction after functional treatment of acute lateral ankle injuries at emergency departments versus family doctor offices

BACKGROUND: In some Western countries, more and more patients seek initial treatment even for minor injuries at emergency units of hospitals. The initial evaluation and treatment as well as aftercare of these patients require large amounts of personnel and logistical resources, which are limited and costly, especially if compared to treatment by a general practitioner. In this study, we investigated whether outsourcing from our level 1 trauma center to a general practitioner has an influence on patient satisfaction and compliance. METHODS: This prospective, randomized study, included n = 100 patients who suffered from a lateral ankle ligament injury grade I-II (16, 17). After radiological exclusion of osseous lesions, the patients received early functional treatment and were shown physical therapy exercises to be done at home, without immobilization or the use of stabilizing ortheses. The patients were randomly assigned into two groups of 50 patients each: Group A (ER): Follow-up and final examination in the hospital's emergency unit. Group B (GP): Follow-up by general practitioner, final examination at hospital's emergency unit. The patients were surveyed regarding their satisfaction with the treatment and outcome of the treatment. RESULTS: Female and male patients were equally represented in both groups. The age of the patients ranged from 16 - 64 years, with a mean age of 34 years (ER) and 35 years (GP). 98% (n = 98) of all patients were satisfied with their treatment, and 93% (n = 93) were satisfied with the outcome. For these parameters no significant difference between the two groups could be noted (p = 0.7406 and 0.7631 respectively). 39% of all patients acquired stabilizing ortheses like ankle braces (Aircast, Malleoloc etc.) on their own initiative. There was a not significant tendency for more self-acquired ortheses in the group treated by general practicioners (p = 0,2669). CONCLUSION: Patients who first present at the ER with a lateral ankle ligament injury grade I-II can be referred to a general practitioner for follow-up treatment without affecting patient satisfaction regarding treatment and treatment outcome.

Pregnancy induces numerical and functional changes of CD4+CD25 high regulatory T cells in patients with rheumatoid arthritis

OBJECTIVE: In a prospective study we investigated whether numerical and functional changes of CD4+CD25(high) regulatory T cells (Treg) were associated with changes of disease activity observed during pregnancy and post partum in patients with rheumatoid arthritis (RA). METHODS: The frequency of CD4+CD25(high) T cells was determined by flow cytometry in 12 patients with RA and 14 healthy women during and after pregnancy. Fluorescence-activated cell sorting (FACS) was used to sort CD4+CD25(high) T cells and CD4+CD25- T cells were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies alone or in co-culture to investigate proliferation and cytokine secretion. RESULTS: Frequencies of CD4+CD25(high) Treg were significantly higher in the third trimester compared to 8 weeks post partum in patients and controls. Numbers of CD4+CD25(high) Treg inversely correlated with disease activity in the third trimester and post partum. In co-culture experiments significantly higher amounts of IL10 and lowered levels of tumour necrosis factor (TNF)alpha and interferon (IFN)gamma were found in supernatants of the third trimester compared to postpartum samples. These findings were independent from health or disease in pregnancy, however postpartum TNFalpha and IFN gamma levels were higher in patients with disease flares. CONCLUSION: The amelioration of disease activity in the third trimester corresponded to the increased number of Treg that induced a pronounced anti-inflammatory cytokine milieu. The pregnancy related quantitative and qualitative changes of Treg suggest a beneficial effect of Treg on disease activity.

Functional effect of FGF2- and FGF8-expanded ventral mesencephalic precursor cells in a rat model of Parkinson's disease

Ventral mesencephalic (VM) precursor cells are of interest in the search for transplantable dopaminergic neurons for cell therapy in Parkinson's disease (PD). In the present study we investigated the survival and functional capacity of in vitro expanded, primary VM precursor cells after intrastriatal grafting to a rat model of PD. Embryonic day 12 rat VM tissue was mechanically dissociated and cultured for 4 or 8 days in vitro (DIV) in the presence of FGF2 (20 ng/ml), FGF8 (20 ng/ml) or without mitogens (control). Cells were thereafter differentiated for 6 DIV by mitogen withdrawal and addition of serum. After differentiation, significantly more tyrosine hydroxylase-immunoreactive (TH-ir), dopamine-producing neurons were found in FGF2- and FGF8-expanded cultures compared to controls. Moreover, expansion for 4 DIV resulted in significantly more TH-ir cells than expansion for 8 DIV both for FGF2 (2.4 fold; P<0.001) and FGF8 (3.8 fold; P<0.001) treated cultures. The functional potential of the expanded cells (4 DIV) was examined after grafting into striatum of aged 6-hydroxydopamine-lesioned rats. Amphetamine-induced rotations performed 3, 6 and 9 weeks postgrafting revealed that grafts of FGF2-expanded cells induced a significantly faster and better functional recovery than grafts of FGF8-expanded cells or control cells (P<0.05 for both). Grafts of FGF2-expanded cells also contained significantly more TH-ir cells than grafts of FGF8-expanded cells (P<0.05) or control cells (P<0.01). In conclusion, FGF2-mediated pregrafting expansion of primary VM precursor cells considerably improves dopaminergic cell survival and functional restoration in a rat model of PD.

The complement inhibitor low molecular weight dextran sulfate prevents TLR4-induced phenotypic and functional maturation of human dendritic cells

Low molecular weight dextran sulfate (DXS) has been reported to inhibit the classical, alternative pathway as well as the mannan-binding lectin pathway of the complement system. Furthermore, it acts as an endothelial cell protectant inhibiting complement-mediated endothelial cell damage. Endothelial cells are covered with a layer of heparan sulfate (HS), which is rapidly released under conditions of inflammation and tissue injury. Soluble HS induces maturation of dendritic cells (DC) via TLR4. In this study, we show the inhibitory effect of DXS on human DC maturation. DXS significantly prevents phenotypic maturation of monocyte-derived DC and peripheral myeloid DC by inhibiting the up-regulation of CD40, CD80, CD83, CD86, ICAM-1, and HLA-DR and down-regulates DC-SIGN in response to HS or exogenous TLR ligands. DXS also inhibits the functional maturation of DC as demonstrated by reduced T cell proliferation, and strongly impairs secretion of the proinflammatory mediators IL-1beta, IL-6, IL-12p70, and TNF-alpha. Exposure to DXS leads to a reduced production of the complement component C1q and a decreased phagocytic activity, whereas C3 secretion is increased. Moreover, DXS was found to inhibit phosphorylation of IkappaB-alpha and activation of NF-kappaB. These findings suggest that DXS prevents TLR-induced maturation of human DC and may therefore be a useful reagent to impede the link between innate and adaptive immunity.

Linking physiology with behaviour: Functional specialisation of the visual field is reflected in gaze patterns during visual search

Based on neurophysiological findings and a grid to score binocular visual field function, two hypotheses concerning the spatial distribution of fixations during visual search were tested and confirmed in healthy participants and patients with homonymous visual field defects. Both groups showed significant biases of fixations and viewing time towards the centre of the screen and the upper screen half. Patients displayed a third bias towards the side of their field defect, which represents oculomotor compensation. Moreover, significant correlations between the extent of these three biases and search performance were found. Our findings suggest a new, more dynamic view of how functional specialisation of the visual field influences behaviour.

Role of glial cells in the functional expression of LL-37/rat cathelin-related antimicrobial peptide in meningitis

Antimicrobial peptides are intrinsic to the innate immune system in many organ systems, but little is known about their expression in the central nervous system. We examined cerebrospinal fluid (CSF) and serum from patients with active bacterial meningitis to assess antimicrobial peptides and possible bactericidal properties of the CSF. We found antimicrobial peptides (human cathelicidin LL-37) in the CSF of patients with bacterial meningitis but not in control CSF. We next characterized the expression, secretion, and bactericidal properties of rat cathelin-related antimicrobial peptide, the homologue of the human LL-37, in rat astrocytes and microglia after incubation with different bacterial components. Using real-time polymerase chain reaction and Western blotting, we determined that supernatants from both astrocytes and microglia incubated with bacterial component supernatants had antimicrobial activity. The expression of rat cathelin-related antimicrobial peptide in rat glial cells involved different signal transduction pathways and was induced by the inflammatory cytokines interleukin 1beta and tumor necrosis factor. In an experimental model of meningitis, infant rats were intracisternally infected with Streptococcus pneumoniae, and rat cathelin-related antimicrobial peptide was localized in glia, choroid plexus, and ependymal cells by immunohistochemistry. Together, these results suggest that cathelicidins produced by glia and other cells play an important part in the innate immune response against pathogens in central nervous system bacterial infections.

Isolation and functional characterization of a stable complex between photoactivated rhodopsin and the G protein, transducin

Transitory binding between photoactivated rhodopsin (Rho* or Meta II) and the G protein transducin (Gt-GDP) is the first step in the visual signaling cascade. Light causes photoisomerization of the 11-cis-retinylidene chromophore in rhodopsin (Rho) to all-trans-retinylidene, which induces conformational changes that allow Gt-GDP to dock onto the Rho* surface. GDP then dissociates from Gt, leaving a transient nucleotide-empty Rho*-Gt(e) complex before GTP becomes bound, and Gt-GTP then dissociates from Rho*. Further biochemical advances are required before structural studies of the various Rho*-Gt complexes can be initiated. Here, we describe the isolation of n-dodecyl-beta-maltoside solubilized, stable, functionally active, Rho*-Gt(e), Rho(e)*-Gt(e), and 9-cis-retinal/11-cis-retinal regenerated Rho-Gt(e) complexes by sucrose gradient centrifugation. In these complexes, Rho* spectrally remained in its Meta II state, and Gt(e) retained its ability to interact with GTPgammaS. Removal of all-trans-retinylidene from Rho*-Gt(e) had no effect on the stability of the Rho(e)*-Gt(e) complex. Moreover, opsin in the Rho(e)*-Gt(e) complex with an empty nucleotide-binding pocket in Gt and an empty retinoid-binding pocket in Rho was regenerated up to 75% without complex dissociation. These results indicate that once Rho* couples with Gt, the chromophore plays a minor role in stabilizing this complex. Moreover, in complexes regenerated with 9-cis-retinal/11-cis-retinal, Rho retains a conformation similar to Rho* that is stabilized by Gt(e) apo-protein.