Primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease with an incidence estimated between 1:2,000 and 1:40,000. Ciliated epithelia line the airways, nasal and sinus cavities, Eustachian tube and fallopian tubes. Congenital abnormalities of ciliary structure and function impair mucociliary clearance. As a consequence, patients present with chronic sinopulmonary infections, recurrent glue ear and female subfertility. Similarities in the ultrastructure of respiratory cilia, nodal cilia and sperm result in patients with PCD also presenting with male infertility, abnormalities of left-right asymmetry (most commonly situs inversus totalis) and congenital heart disease. Early diagnosis is essential to ensure specialist management of the respiratory and otological complications of PCD. Diagnostic tests focus on analysis of ciliary function and electron microscopy structure. Analysis is technically difficult and labour intensive. It requires expertise for interpretation, restricting diagnosis to specialist centres. Management is currently based on the consensus of experts, and there is a pressing need for randomised clinical trials to inform treatment.

Trypanosoma brucei: a model micro-organism to study eukaryotic phospholipid biosynthesis

Although the protozoan parasite, Trypanosoma brucei, can acquire lipids from its environment, recent reports have shown that it is also capable of de novo synthesis of all major phospholipids. Here we provide an overview of the biosynthetic pathways involved in phospholipid formation in T. brucei and highlight differences to corresponding pathways in other eukaryotes, with the aim of promoting trypanosomes as an attractive model organism to study lipid biosynthesis. We show that de novo synthesis of phosphatidylethanolamine involving CDP-activated intermediates is essential in T. brucei and that a reduction in its cellular content affects mitochondrial morphology and ultrastructure. In addition, we highlight that reduced levels of phosphatidylcholine inhibit nuclear division, suggesting a role for phosphatidylcholine formation in the control of cell division. Furthermore, we discuss possible routes leading to phosphatidylserine and cardiolipin formation in T. brucei and review the biosynthesis of phosphatidylinositol, which seems to take place in two separate compartments. Finally, we emphasize that T. brucei represents the only eukaryote so far that synthesizes all three sphingophospholipid classes, sphingomyelin, inositolphosphorylceramide and ethanolaminephosphorylceramide, and that their production is developmentally regulated.

Cardiac tissue-restricted deletion of plakoglobin results in progressive cardiomyopathy and activation of {beta}-catenin signaling

Mutations in the plakoglobin (JUP) gene have been identified in arrhythmogenic right ventricular cardiomyopathy (ARVC) patients. However, the mechanisms underlying plakoglobin dysfunction involved in the pathogenesis of ARVC remain poorly understood. Plakoglobin is a component of both desmosomes and adherens junctions located at the intercalated disc (ICD) of cardiomyocytes, where it functions to link cadherins to the cytoskeleton. In addition, plakoglobin functions as a signaling protein via its ability to modulate the Wnt/beta-catenin signaling pathway. To investigate the role of plakoglobin in ARVC, we generated an inducible cardiorestricted knockout (CKO) of the plakoglobin gene in mice. Plakoglobin CKO mice exhibited progressive loss of cardiac myocytes, extensive inflammatory infiltration, fibrous tissue replacement, and cardiac dysfunction similar to those of ARVC patients. Desmosomal proteins from the ICD were decreased, consistent with altered desmosome ultrastructure in plakoglobin CKO hearts. Despite gap junction remodeling, plakoglobin CKO hearts were refractory to induced arrhythmias. Ablation of plakoglobin caused increase beta-catenin stabilization associated with activated AKT and inhibition of glycogen synthase kinase 3beta. Finally, beta-catenin/TCF transcriptional activity may contribute to the cardiac hypertrophy response in plakoglobin CKO mice. This novel model of ARVC demonstrates for the first time how plakoglobin affects beta-catenin activity in the heart and its implications for disease pathogenesis.

MAP kinase kinase 1 (MKK1) is essential for transmission of Trypanosoma brucei by Glossina morsitans

MAP kinase kinase 1 (MKK1) is encoded by a single copy gene in Trypanosoma brucei. It has been shown recently that MKK1 is not essential for bloodstream forms [14]. To investigate the requirement for MKK1 in other life-cycle stages we generated null mutants in procyclic forms of a fly-transmissible strain. These grew normally in culture and were able to establish midgut infections in tsetse at normal rates and intensities, but were incapable of colonising the salivary glands. Transformation of null mutants with an ectopic copy of MKK1 enabled parasites to complete the life cycle in tsetse and infect mice. This is the first example of a gene that is indispensable for transmission of T. brucei. It also raises the possibility that activating the MKK1 signalling cascade in vitro might trigger the differentiation and proliferation of life-cycle stages of T. brucei that are currently refractory to culture.

Valve disease in chronic venous disorders: a quantitative ultrastructural analysis by transmission electron microscopy and stereology

INTRODUCTION: The ultrastructure of venous valves and walls in chronic venous disease was investigated. METHODS: Consecutive patients were categorised into one of three groups (group A: patients with C1 venous disease in accordance with CEAP (Clinical severity, Etiology, Anatomy, Pathophysiology); group B: C2 and C3; group C: C4, C5 and C6). The terminal or preterminal valve and adjacent vessel wall was harvested from the great saphenous vein. Sections were examined with a transmission electron microscope. The volumes of elastin and of collagen per unit surface area of valve were assessed, as well as the surface endothelium of valve and vessel wall. RESULTS: The study population consisted of 17 patients. The elastin ratio was analysed by means of stereology. Mean values were: in group A, 0.45 μm3/m2; in group B, 0.67 μm3/m2; in group C, 0.97 μm3/m2. The ratio was similar for collagen (A, 15.7 μm3/m2; B, 26.8 μm3/m2; C, 30.1 μm3/m2). Surface analysis of the valve endothelium and the adjacent vessel wall endothelium showed a trend towards increasing damage with more severe disease. CONCLUSIONS: With progression of venous disease, the valve elastin content, assessed morphologically, seems to increase, and the endothelium of the venous valve and the vein wall tend to show more damage.

Black hair follicular dysplasia in Large Münsterländer dogs: clinical, histological and ultrastructural features

Four Large Münsterländer cross-bred dogs affected with black hair follicular dysplasia (BHFD) and one unaffected control littermate were observed, and skin was sampled weekly over the first 19 weeks of life. Affected dogs were born with silvery grey hair, a consequence of melanin clumping in the hair shafts. Hair bulb melanocytes were densely pigmented, and contained abundant stage IV melanosomes but adjacent matrix keratinocytes lacked melanosomes. Melanin clumping was not prominent in epidermal melanocytes in the haired skin but occurred in the foot pads. Follicular changes progressed from bulbar clumping, clumping in the isthmus/infundibulum and finally to dysplastic hair shafts. Alopecia developed progressively in pigmented areas. Silver-grey hair, melanin clumping, accumulation of stage IV melanosomes within melanocytes and insufficient melanin transfer to adjacent keratinocytes are also classic features of human Griscelli syndrome. The underlying cause in Griscelli syndrome is a defect of melanocytic intracellular transport proteins leading to inadequate and disorganized melanosome transfer to keratinocytes with resultant melanin clumping. In view of the correlation in the phenotype, histology and ultrastructure between both disorders, a defect in intracellular melanosome transport is postulated as the pathogenic mechanism in BHFD.

Trypanosoma congolense procyclins: unmasking cryptic major surface glycoproteins in procyclic forms

In the tsetse fly, the protozoan parasite Trypanosoma congolense is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These include a protease-resistant surface molecule (PRS), which is expressed by procyclic forms early in infection, and a glutamic acid- and alanine-rich protein (GARP), which appears at later stages. Since neither of these surface antigens is expressed at intermediate stages, we investigated whether a GPI-anchored protein of 50 to 58 kDa, previously detected in procyclic culture forms, might constitute the coat of these parasites. We therefore partially purified the protein from T. congolense Kilifi procyclic forms, obtained an N-terminal amino acid sequence, and identified its gene. Detailed analyses showed that the mature protein consists almost exclusively of 13 heptapeptide repeats (EPGENGT). The protein is densely N glycosylated, with up to 13 high-mannose oligosaccharides ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) linked to the peptide repeats. The lipid moiety of the glycosylphosphatidylinositol is composed of sn-1-stearoyl-2-lyso-glycerol-3-HPO(4)-1-(2-O-acyl)-d-myo-inositol. Heavily glycosylated proteins with similar repeats were subsequently identified in T. congolense Savannah procyclic forms. Collectively, this group of proteins was named T. congolense procyclins to reflect their relationship to the EP and GPEET procyclins of T. brucei. Using an antiserum raised against the EPGENGT repeat, we show that T. congolense procyclins are expressed continuously in the fly midgut and thus form the surface coat of cells that are negative for both PRS and GARP.

Treatment with (90)Y- and (177)Lu-DOTATOC in patients with metastatic neuroendocrine tumors

BACKGROUND: Treatment with (90)Y- or (177)Lu-DOTATOC has recently been introduced in the palliative treatment of somatostatin receptor-expressing neuroendocrine tumors (NETs). The aim of the study was to present clinical experience with (90)Y- and (177)Lu-DOTATOC therapy in the management of NET. METHODS: To prove suitability for treatment each patient underwent scanning with (111)In-DTPAOC or (68)Ga-DOTATOC positron emission tomography/computed tomography. All patients received [(90)Y-DOTATOC] as initial treatment. In case of disease relapse the treatment was repeated. To avoid side effects of repeated [(90)Y] applications, a switch to [(177)Lu-DOTATOC] was carried out. Clinical, biochemical, and radioimaging responses were documented. RESULTS: Twenty patients with metastatic nonresectable NETs (15 pancreas NETs, 2 midgut NETs, 1 gastrinoma, 1 paraganglioma, 1 NET of unknown primary origin) were included. In 8 patients the treatment was repeated more than once (mean, 3 times; range, 2-5 times). After [(90)Y] treatment moderate toxicity was observed in 8 patients. No serious adverse events were documentable. After restaging, a partial remission was found in 5 patients, stable disease in 11 patients, and tumor progression in 4 patients. CONCLUSIONS: Peptide receptor-targeted radionuclide therapy is a promising, safe, and feasible approach in the palliative therapy of patients with NET.

New ways of looking at synapses

Current concepts of synaptic fine-structure are derived from electron microscopic studies of tissue fixed by chemical fixation using aldehydes. However, chemical fixation with glutaraldehyde and paraformaldehyde and subsequent dehydration in ethanol result in uncontrolled tissue shrinkage. While electron microscopy allows for the unequivocal identification of synaptic contacts, it cannot be used for real-time analysis of structural changes at synapses. For the latter purpose advanced fluorescence microscopy techniques are to be applied which, however, do not allow for the identification of synaptic contacts. Here, two approaches are described that may overcome, at least in part, some of these drawbacks in the study of synapses. By focusing on a characteristic, easily identifiable synapse, the mossy fiber synapse in the hippocampus, we first describe high-pressure freezing of fresh tissue as a method that may be applied to study subtle changes in synaptic ultrastructure associated with functional synaptic plasticity. Next, we propose to label presynaptic mossy fiber terminals and postsynaptic complex spines on CA3 pyramidal neurons by different fluorescent dyes to allow for the real-time monitoring of these synapses in living tissue over extended periods of time. We expect these approaches to lead to new insights into the structure and function of central synapses.

Abnormal collagen fibrils in cartilage of matrilin-1/matrilin-3-deficient mice

Matrilins are oligomeric extracellular matrix adaptor proteins mediating interactions between collagen fibrils and other matrix constituents. All four matrilins are expressed in cartilage and mutations in the human gene encoding matrilin-3 (MATN3) are associated with different forms of chondrodysplasia. Surprisingly, however, Matn3-null as well as Matn1- and Matn2-null mice do not show an overt skeletal phenotype, suggesting a dominant negative pathomechanism for the human disorders and redundancy/compensation among the family members in the knock-out situation. Here, we show that mice lacking both matrilin-1 and matrilin-3 develop an apparently normal skeleton, but exhibit biochemical and ultrastructural abnormalities of the knee joint cartilage. At the protein level, an altered SDS-PAGE band pattern and a clear up-regulation of the homotrimeric form of matrilin-4 were evident in newborn Matn1/Matn3 and Matn1 knock-out mice, but not in Matn3-null mice. The ultrastructure of the cartilage matrix after conventional chemical fixation was grossly normal; however, electron microscopy of high pressure frozen and freeze-substituted samples, revealed two consistent observations: 1) moderately increased collagen fibril diameters throughout the epiphysis and the growth plate in both single and double mutants; and 2) increased collagen volume density in Matn1(-/-)/Matn3(-/-) and Matn3(-/-) mice. Taken together, our results demonstrate that matrilin-1 and matrilin-3 modulate collagen fibrillogenesis in cartilage and provide evidence that biochemical compensation might exist between matrilins.

A family of stage-specific alanine-rich proteins on the surface of epimastigote forms of Trypanosoma brucei

A 'two coat' model of the life cycle of Trypanosoma brucei has prevailed for more than 15 years. Metacyclic forms transmitted by infected tsetse flies and mammalian bloodstream forms are covered by variant surface glycoproteins. All other life cycle stages were believed to have a procyclin coat, until it was shown recently that epimastigote forms in tsetse salivary glands express procyclin mRNAs without translating them. As epimastigote forms cannot be cultured, a procedure was devised to compare the transcriptomes of parasites in different fly tissues. Transcripts encoding a family of glycosylphosphatidyl inositol-anchored proteins, BARPs (previously called bloodstream alanine-rich proteins), were 20-fold more abundant in salivary gland than midgut (procyclic) trypanosomes. Anti-BARP antisera reacted strongly and exclusively with salivary gland parasites and a BARP 3' flanking region directed epimastigote-specific expression of reporter genes in the fly, but inhibited expression in bloodstream and procyclic forms. In contrast to an earlier report, we could not detect BARPs in bloodstream forms. We propose that BARPs form a stage-specific coat for epimastigote forms and suggest renaming them brucei alanine-rich proteins.

Effect of GH on human skeletal muscle lipid metabolism in GH deficiency

Adult-onset growth hormone (GH) deficiency (GHD) is associated with insulin resistance and decreased exercise capacity. Intramyocellular lipids (IMCL) depend on training status, diet, and insulin sensitivity. Using magnetic resonance spectroscopy, we studied IMCL content following physical activity (IMCL-depleted) and high-fat diet (IMCL-repleted) in 15 patients with GHD before and after 4 mo of GH replacement therapy (GHRT) and in 11 healthy control subjects. Measurements of insulin resistance and exercise capacity were performed and skeletal muscle biopsies were carried out to assess expression of mRNA of key enzymes involved in skeletal muscle lipid metabolism by real-time PCR and ultrastructure by electron microscopy. Compared with control subjects, patients with GHD showed significantly higher difference between IMCL-depleted and IMCL-repleted. GHRT resulted in an increase in skeletal muscle mRNA expression of IGF-I, hormone-sensitive lipase, and a tendency for an increase in fatty acid binding protein-3. Electron microscopy examination did not reveal significant differences after GHRT. In conclusion, variation of IMCL may be increased in patients with GHD compared with healthy control subjects. Qualitative changes within the skeletal muscle (i.e., an increase in free fatty acids availability from systemic and/or local sources) may contribute to the increase in insulin resistance and possibly to the improvement of exercise capacity after GHRT. The upregulation of IGF-I mRNA suggests a paracrine/autocrine role of IGF-I on skeletal muscle.

Exogenous surfactant application in a rat lung ischemia reperfusion injury model: effects on edema formation and alveolar type II cells

BACKGROUND: Prophylactic exogenous surfactant therapy is a promising way to attenuate the ischemia and reperfusion (I/R) injury associated with lung transplantation and thereby to decrease the clinical occurrence of acute lung injury and acute respiratory distress syndrome. However, there is little information on the mode by which exogenous surfactant attenuates I/R injury of the lung. We hypothesized that exogenous surfactant may act by limiting pulmonary edema formation and by enhancing alveolar type II cell and lamellar body preservation. Therefore, we investigated the effect of exogenous surfactant therapy on the formation of pulmonary edema in different lung compartments and on the ultrastructure of the surfactant producing alveolar epithelial type II cells. METHODS: Rats were randomly assigned to a control, Celsior (CE) or Celsior + surfactant (CE+S) group (n = 5 each). In both Celsior groups, the lungs were flush-perfused with Celsior and subsequently exposed to 4 h of extracorporeal ischemia at 4 degrees C and 50 min of reperfusion at 37 degrees C. The CE+S group received an intratracheal bolus of a modified natural bovine surfactant at a dosage of 50 mg/kg body weight before flush perfusion. After reperfusion (Celsior groups) or immediately after sacrifice (Control), the lungs were fixed by vascular perfusion and processed for light and electron microscopy. Stereology was used to quantify edematous changes as well as alterations of the alveolar epithelial type II cells. RESULTS: Surfactant treatment decreased the intraalveolar edema formation (mean (coefficient of variation): CE: 160 mm3 (0.61) vs. CE+S: 4 mm3 (0.75); p < 0.05) and the development of atelectases (CE: 342 mm3 (0.90) vs. CE+S: 0 mm3; p < 0.05) but led to a higher degree of peribronchovascular edema (CE: 89 mm3 (0.39) vs. CE+S: 268 mm3 (0.43); p < 0.05). Alveolar type II cells were similarly swollen in CE (423 microm3(0.10)) and CE+S (481 microm3(0.10)) compared with controls (323 microm3(0.07); p < 0.05 vs. CE and CE+S). The number of lamellar bodies was increased and the mean lamellar body volume was decreased in both CE groups compared with the control group (p < 0.05). CONCLUSION: Intratracheal surfactant application before I/R significantly reduces the intraalveolar edema formation and development of atelectases but leads to an increased development of peribronchovascular edema. Morphological changes of alveolar type II cells due to I/R are not affected by surfactant treatment. The beneficial effects of exogenous surfactant therapy are related to the intraalveolar activity of the exogenous surfactant.

Initial results of in vivo high-resolution morphological and biochemical cartilage imaging of patients after matrix-associated autologous chondrocyte transplantation (MACT) of the ankle

OBJECTIVE: The aim of this study was to use morphological as well as biochemical (T2 and T2* relaxation times and diffusion-weighted imaging (DWI)) magnetic resonance imaging (MRI) for the evaluation of healthy cartilage and cartilage repair tissue after matrix-associated autologous chondrocyte transplantation (MACT) of the ankle joint. MATERIALS AND METHODS: Ten healthy volunteers (mean age, 32.4 years) and 12 patients who underwent MACT of the ankle joint (mean age, 32.8 years) were included. In order to evaluate possible maturation effects, patients were separated into short-term (6-13 months) and long-term (20-54 months) follow-up cohorts. MRI was performed on a 3.0-T magnetic resonance (MR) scanner using a new dedicated eight-channel foot-and-ankle coil. Using high-resolution morphological MRI, the magnetic resonance observation of cartilage repair tissue (MOCART) score was assessed. For biochemical MRI, T2 mapping, T2* mapping, and DWI were obtained. Region-of-interest analysis was performed within native cartilage of the volunteers and control cartilage as well as cartilage repair tissue in the patients subsequent to MACT. RESULTS: The overall MOCART score in patients after MACT was 73.8. T2 relaxation times (approximately 50 ms), T2* relaxation times (approximately 16 ms), and the diffusion constant for DWI (approximately 1.3) were comparable for the healthy volunteers and the control cartilage in the patients after MACT. The cartilage repair tissue showed no significant difference in T2 and T2* relaxation times (p > or = 0.05) compared to the control cartilage; however, a significantly higher diffusivity (approximately 1.5; p < 0.05) was noted in the cartilage repair tissue. CONCLUSION: The obtained results suggest that besides morphological MRI and biochemical MR techniques, such as T2 and T2* mapping, DWI may also deliver additional information about the ultrastructure of cartilage and cartilage repair tissue in the ankle joint using high-field MRI, a dedicated multichannel coil, and sophisticated sequences.

Perturbation of phosphatidylethanolamine synthesis affects mitochondrial morphology and cell-cycle progression in procyclic-form Trypanosoma brucei

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are the two major constituents of eukaryotic cell membranes. In the protist Trypanosoma brucei, PE and PC are synthesized exclusively via the Kennedy pathway. To determine which organelles or processes are most sensitive to a disruption of normal phospholipid levels, the cellular consequences of a decrease in the levels of PE or PC, respectively, were studied following RNAi knock-down of four enzymes of the Kennedy pathway. RNAi against ethanolamine-phosphate cytidylyltransferase (ET) disrupted mitochondrial morphology and ultrastructure. Electron microscopy revealed alterations of inner mitochondrial membrane morphology, defined by a loss of disk-like cristae. Despite the structural changes in the mitochondrion, the cells maintained oxidative phosphorylation. Our results indicate that the inner membrane morphology of T. brucei procyclic forms is highly sensitive to a decrease of PE levels, as a change in the ultrastructure of the mitochondrion is the earliest phenotype observed after RNAi knock-down of ET. Interference with phospholipid synthesis also impaired normal cell-cycle progression. ET RNAi led to an accumulation of multinucleate cells. In contrast, RNAi against choline-/ethanolamine phosphotransferase, which affected PC as well as PE levels, caused a cell division phenotype characterized by non-division of the nucleus and production of zoids.