Advanced gelatin-based vascularization bioinks for extrusion-based bioprinting of vascularized bone equivalents.

Bone tissue is highly vascularized. The crosstalk of vascular and osteogenic cells is not only responsible for the formation of the strongly divergent tissue types but also for their physiological maintenance and repair. Extrusion-based bioprinting presents a promising fabrication method for bone replacement. It allows for the production of large-volume constructs, which can be tailored to individual tissue defect geometries. In this study, we used the all-gelatin-based toolbox of methacryl-modified gelatin (GM), non-modified gelatin (G) and acetylated GM (GMA) to tailor both the properties of the bioink towards improved printability, and the properties of the crosslinked hydrogel towards enhanced support of vascular network formation by simple blending. The vasculogenic behavior of human dermal microvascular endothelial cells (HDMECs) and human adipose-derived stem cells (ASCs) was evaluated in the different hydrogel formulations for 14 days. Co-culture constructs including a vascular component and an osteogenic component (i.e. a bone bioink based on GM, hydroxyapatite and ASCs) were fabricated via extrusion-based bioprinting. Bioprinted co-culture constructs exhibited functional tissue-specific cells whose interplay positively affected the formation and maintenance of vascular-like structures. The setup further enabled the deposition of bone matrix associated proteins like collagen type I, fibronectin and alkaline phosphatase within the 30-day culture.

Extended surgical safety margins and ulceration are associated with an improved prognosis in pleomorphic dermal sarcomas.

BACKGROUND: Pleomorphic dermal sarcomas (PDS) are frequent UV-induced sarcomas of the skin of intermediate grade malignant potential. Despite the fact that PDS have a noteworthy potential to recur (up to 28%) as well as to metastasize (up to 20%), there are no specific clinical guidelines with respect to follow-up these patients. Moreover, little is known about clinical, histological or molecular prognostic factors in PDS. OBJECTIVE: The aim of the present study was to identify risk factors to predict relapse in a large multicentre sample cohort of PDS which could aid to optimize personalized treatment recommendations regarding surgical safety margins and adjuvant radiotherapy. METHODS: Patients with a diagnosis of PDS were selected from nine European institutions based on the histopathologic criteria described by Fletcher. Clinicopathologic and follow-up data were collected and statistically analysed calculating univariate hazard ratios with 95% confidence intervals by use of the Cox proportional-hazards model and a significance level of P < 0.05. Patients with an incomplete excision of the tumour were excluded. RESULTS: Univariate Cox regression analysis of possible prognostic factors for progression-free survival (PFS) performed in 92 patients revealed that an excision margin of <2 cm is significantly associated with relapse of PDS [hazard ratio 4.478 (95% CI 1.536-13.055), P = 0.006]. Ulceration of the tumour was associated with a significantly better prognosis [0.396 (0.174-0.904), P = 0.028] whereas adjuvant radiotherapy did not reach statistical significance to improve prognosis in patients with PDS [0.775 (0.231-2.593), P = 0.679]. Gender, age, immunosuppression, intratumoural necrosis, tumour location, vertical thickness or horizontal diameter did not significantly influence PFS in PDS. CONCLUSION: We identified surgical safety margins of <2 cm and absence of ulceration as risk factors for relapse in patients with PDS. These findings may be implemented into both the primary treatment as well as the further monitoring of patients with PDS.

Modified Vaccinia Virus Ankara: History, Value in Basic Research, and Current Perspectives for Vaccine Development.

Safety tested Modified Vaccinia virus Ankara (MVA) is licensed as third-generation vaccine against smallpox and serves as a potent vector system for development of new candidate vaccines against infectious diseases and cancer. Historically, MVA was developed by serial tissue culture passage in primary chicken cells of vaccinia virus strain Ankara, and clinically used to avoid the undesirable side effects of conventional smallpox vaccination. Adapted to growth in avian cells MVA lost the ability to replicate in mammalian hosts and lacks many of the genes orthopoxviruses use to conquer their host (cell) environment. As a biologically well-characterized mutant virus, MVA facilitates fundamental research to elucidate the functions of poxvirus host-interaction factors. As extremely safe viral vectors MVA vaccines have been found immunogenic and protective in various preclinical infection models. Multiple recombinant MVA currently undergo clinical testing for vaccination against human immunodeficiency viruses, Mycobacterium tuberculosis or Plasmodium falciparum. The versatility of the MVA vector vaccine platform is readily demonstrated by the swift development of experimental vaccines for immunization against emerging infections such as the Middle East Respiratory Syndrome. Recent advances include promising results from the clinical testing of recombinant MVA-producing antigens of highly pathogenic avian influenza virus H5N1 or Ebola virus. This review summarizes our current knowledge about MVA as a unique strain of vaccinia virus, and discusses the prospects of exploiting this virus as research tool in poxvirus biology or as safe viral vector vaccine to challenge existing and future bottlenecks in vaccinology.

[Prevalence of glaucoma in obstructive sleep apnea]. / Glaukomprävalenz bei obstruktiver Schlafapnoe.

BACKGROUND: A causal relationship between glaucoma and obstructive sleep apnea has been postulated in several clinical studies but also refuted by others. The aim of this study was to determine the prevalence of glaucoma in a cohort of patients with well-established obstructive sleep apnea in comparison to the published data on this topic. METHODS: A total of 100 consecutive patients (male:female 80:20, mean age 59 ± 11 years SD) with polysomnographically established obstructive sleep apnea underwent an ophthalmological examination including tonometry, static perimetry and dilated fundus photography. Visual fields and fundus photographs of the patients were classified as glaucomatous or non-glaucomatous by two independent examiners. RESULTS: The prevalence of glaucoma in the study patients was 2 % which corresponded to the published prevalence of glaucoma in the normal population. Intraocular pressure did not correlate with the respiratory index, body mass index or sex. CONCLUSION: The data from this study shed doubt on a causal relationship between obstructive sleep apnea and glaucoma.

Verrucous carcinoma in epidermolysis bullosa simplex is possibly associated with a novel mutation in the keratin 5 gene.

Epidermolysis bullosa simplex (EBS) is mainly caused by mutations in the KRT5 and KRT14 genes. Squamous cell carcinoma (SCC) represents the second most frequent skin neoplasia with complex aetiology. The molecular events disrupting the orchestrated interplay between the cytoskeleton, cell adhesion molecules and signalling proteins are ill understood in SCC. We describe the molecular background and the unusual course of the disease in a patient with EBS Dowling-Meara, severe keratoderma and a massive verrucous carcinoma. Skin and tumour samples from the patient were analysed using light microscopy, immunohistochemistry and immunofluorescence mapping. Mutation analysis of the KRT5 and KRT14 genes identified the novel KRT5 mutation p.E477D. Invasive tumour areas were characterized by downregulation of keratins 5 and 14, reduced and irregular desmocollin-2 expression and increased expression of keratins 6, 16 and 17. Levels of Ki-67 were increased and levels of E-cadherin strongly reduced in the tumour tissue. In this case a novel KRT5 mutation led to increased fragility of keratinocytes. Desmosome and adherens junctions were destabilized, which may trigger keratinocyte-mediated inflammation, possibly via p120-catenin-dependent signalling, suggesting a link between a keratin mutation and SCC, which adds weight to the hypothesis that disturbance of the cytoskeleton represents a major cause in the appearance of the malignant phenotype. Some individuals with EBS may be at risk of developing secondary SCC.

Lethal junctional epidermolysis bullosa with pyloric atresia due to compound heterozygosity for two novel mutations in the integrin ß4 gene.

BACKGROUND: Junctional epidermolysis bullosa with pyloric atresia (JEB-PA) is a rare autosomal recessive disease with blister formation within the lamina lucida due to mutations in the integrin ß4 (ITGB4) and α6 (ITGA6) genes. CASE REPORT: A female preterm infant, first child of healthy non-consanguineous parents, was born at 26 + 4 weeks of gestation by caesarean section, following polyhydramnion and abruption of placenta. She presented with extensive areas of denuded skin on both lateral sides of the head, neck and extremities. Auricles were hypoplastic. Abdominal ultrasound and X-ray were suggestive of pyloric atresia which was revised surgically on the 4th day of life. Further course was complicated by progressive skin detachment, sepsis, and renal insufficiency with fatal outcome at 18 days of age. Immunofluorescence mapping of cryopreserved skin showed junctional cleft formation with negative staining for integrin α6 and integrin ß4. Mutational analysis disclosed compound heterozygosity for two novel nonsense mutations in the ITGB4 gene: c.600dupC/p.F201fsX14 and c.2533C>T/p.Q845X. 2 subsequent pregnancies were terminated following prenatal diagnosis disclosing the same ITGB4 mutations, a 4th pregnancy was unaffected. CONCLUSION: We describe a case of lethal JEB-PA with negative immunoreactivity to integrin α6 and integrin ß4 predicting a poor outcome. Identification of compound heterozygosity for two novel ITGB4 mutations in the affected preterm infant permitted prenatal diagnosis and finally birth of a healthy sibling.

Mild clinical phenotype of Kindler syndrome associated with late diagnosis and skin cancer.

Kindler syndrome (KS) is a heritable skin disorder with a complex phenotype consisting of congenital skin blistering, photosensitivity, progressive generalized poikiloderma and extensive skin atrophy. Here we describe 2 siblings with KS, who are, to the best of our knowledge, the oldest patients reported so far in the literature. The diagnosis was established in their seventh and eighth decades of life, and confirmed by mutation analysis. Both patients were homozygous for the recurrent FERMT1 mutation, c.328C→T, p.R110X. Because of a relatively mild course of the disease, mucosal membranes in the eyes and oesophagus being predominantly affected in recent years, they had been treated under other diagnoses, such as scleroderma. Cutaneous precancerous lesions and epithelial skin cancer arose in both siblings after the age of 50 years and were treated in an early stage. Taken together, we describe the natural course of KS, the morphological abnormalities occurring in the skin of older KS patients, we discuss the differential diagnosis and the association between KS and squamous cell carcinoma.

Postnatal analysis of the effect of embryonic knockdown and overexpression of candidate dyslexia susceptibility gene homolog Dcdc2 in the rat.

Embryonic knockdown of candidate dyslexia susceptibility gene (CDSG) homologs in cerebral cortical progenitor cells in the rat results in acute disturbances of neocortical migration. In the current report we investigated the effects of embryonic knockdown and overexpression of the homolog of DCDC2, one of the CDSGs, on the postnatal organization of the cerebral cortex. Using a within-litter design, we transfected cells in rat embryo neocortical ventricular zone around embryonic day (E) 15 with either 1) small hairpin RNA (shRNA) vectors targeting Dcdc2, 2) a DCDC2 overexpression construct, 3) Dcdc2 shRNA along with DCDC2 overexpression construct, 4) an overexpression construct composed of the C terminal domain of DCDC2, or 5) an overexpression construct composed of the DCX terminal domain of DCDC2. RNAi of Dcdc2 resulted in pockets of heterotopic neurons in the periventricular region. Approximately 25% of the transfected brains had hippocampal pyramidal cell migration anomalies. Dcdc2 shRNA-transfected neurons migrated in a bimodal pattern, with approximately 7% of the neurons migrating a short distance from the ventricular zone, and another 30% migrating past their expected lamina. Rats transfected with Dcdc2 shRNA along with the DCDC2 overexpression construct rescued the periventricular heterotopia phenotype, but did not affect the percentage of transfected neurons that migrate past their expected laminar location. There were no malformations associated with any of the overexpression constructs, nor was there a significant laminar disruption of migration. These results support the claim that knockdown of Dcdc2 expression results in neuronal migration disorders similar to those seen in the brains of dyslexics.

Perforation of the left atrium by a chest tube in a patient with cardiomegaly: management of a rare, but life-threatening complication.

Perforation of the heart is a rare, but life-threatening complication of chest tube thoracostomy. We report the very unusual case where right-sided insertion of a Matthys catheter (6 F) due to pleural effusion resulted in a left atrium perforation. Heart injury was immediately considered as a continuous flow of bright red blood emerging through the chest drain. Diagnosis was confirmed by computertomography also revealing a massive cardiomegaly due to pre-existing mitral valve regurgitation. In two consecutive thoracotomies, first the Mathys drain was removed and the heart defect closed and then the mitral valve was replaced by a bio prosthesis. The extent of the cardiomegaly and the position of the left atrium were not detected pre-operatively by chest X-ray or ultrasonic device. Despite a nosocomial pneumonia, the patient fully recovered. This case shows that extreme caution is necessary when inserting chest tubes in patients where thorax imaging by X-ray or ultrasonic device does not provide a clear anatomical site. In order to minimise complications, a blunt puncturing procedure or Seldinger technique should be used and assisted by a Doppler ultrasonic device. Also early imaging by CT and Doppler ultrasonic technique should be attempted. This may reduce incidence of severe complications as in this case.

Benzodiazepines for schizophrenia.

BACKGROUND: Many people with schizophrenia do not achieve a satisfactory treatment response with ordinary antipsychotic drug treatment. In these cases, various add-on medications are used, among them benzodiazepines. OBJECTIVES: To review the effects of benzodiazepines for the treatment of schizophrenia and schizophrenia-like psychoses. SEARCH STRATEGY: The reviewers searched the Cochrane Schizophrenia Group's register (last search March 2005). This register is compiled by methodical searches of BIOSIS, CINAHL, Dissertation abstracts, EMBASE, LILACS, MEDLINE, PSYNDEX, PsycINFO, RUSSMED, Sociofile, supplemented with hand searching of relevant journals and numerous conference proceedings. We also contacted authors of relevant studies in order to obtain missing data from existing trials. SELECTION CRITERIA: All randomised controlled trials comparing benzodiazepine to antipsychotics or to placebo (or no intervention), whether as sole treatment or as an adjunct to antipsychotic medication for the treatment of schizophrenia and/or schizophrenia-like psychoses. DATA COLLECTION AND ANALYSIS: We independently inspected abstracts, selected studies and re-inspected and quality assessed the full reports. We independently extracted relevant outcomes. Dichotomous data were analysed using relative risks (RR) and the 95% confidence intervals (CI). Continuous data were analysed using weighted mean differences. Where possible the number needed to treat (NNT) or number needed to harm (NNH) statistics were calculated. MAIN RESULTS: The review currently includes 31 studies with over 2000 participants. Most studies were small, of short duration - one to 13 weeks - and inconsistently and incompletely reported. Eight studies compared benzodiazepines as a sole agent with placebo. More participants receiving benzodiazepines showed a clinically significant response (n=222, 4 RCTs, RR 0.54 CI 0.3 to 1.0, NNT 3 CI 2 to 17). Only one small study found a significant group difference in favour of benzodiazepines regarding the improvement in overall BPRS mental state. Different rating scales were used to assess general mental state, and therefore many outcomes could not be pooled and no overall direction of effect emerged. Some adverse events observed in these studies suggested that benzodiazepines were more harmful than placebos but again the data were incompletely reported and without overall effect. Thirteen studies examined the effects of benzodiazepines in comparison to antipsychotics as a sole treatment. Trials that reported on clinical response found no advantage for any treatment group concerning improvement of the participants' global state, except of one small study that analysed the mean CGI severity score at one hour. This comparison is highly limited by the low numbers of studies reporting on global function and the short trial duration. Two studies showed a statistically significant superiority of antipsychotics in terms of relapse prevention at one year. Desired sedation occurred significantly more often among participants in the benzodiazepine group than among participants in the antipsychotic treatment group at 20 (n=301, 1 RCT, RR 1.32 CI 1.2 -1.5, NNT 5, CI 3 to 8) and 40 minutes(n= 301, 1 RCT, RR 1.13 CI 1.0 to 1.2, NNT 9 CI 6 to 33), but not at 30, 60 or 12 minutes. Other outcomes relating to the general or specific mental state revealed no significant differences between groups. As far as adverse events were reported there were no results in favour of any group. Sixteen studies examined whether the augmentation of antipsychotics with benzodiazepines is more effective than antipsychotics as a sole treatment. During the first hour of treatment the combination treatment group benefited from the additional benzodiazepine in terms of the participants global state. This benefit diminished over time and was not reproducible at 2 hours or longer. No superior efficacy of benzodiazepine augmentation could be found regarding the general mental state. Specific aspects of the mental state showed no group difference except for desired sedation at 30 and 60 minutes. Somnolence affected the combination treatment group significantly more than the control group (n=118, 2 RCTs, RR 3.30 CI 1.0 to 10.4, NNH 8 CI 5 to 50). We found use of antiparkinson medication to be less frequently used in the combination treatment group (n=282, RR 0.68 CI 0.5 to 1.0, NNT 9 CI 6 to 48). Adverse events were poorly reported and the results were based on very little data. AUTHORS' CONCLUSIONS: Randomised trial-derived evidence is currently too poor to recommend benzodiazepines neither as a sole nor as an adjunctive agent in schizophrenia or schizophrenia-like psychoses. The only significant effects were seen in terms of short-term sedation, at best. The evidence available on augmentation of antipsychotics with benzodiazepines is inconclusive and justifies large, simple and well-designed future trials focusing on clinical response, mental state, aggressive behaviour and adverse events.

The soluble pool of HLA-G produced by human trophoblasts does not include detectable levels of the intron 4-containing HLA-G5 and HLA-G6 isoforms.

In the context of implantation and pregnancy, several immunomodulating functions have been attributed to the different HLA-G isoforms. Increasing attention is now being addressed to the actively secreted soluble forms, because they might have a systemic function or could be useful as diagnostic tools. However, the cellular source of secretion, even during pregnancy, where HLA-G expression level is known to be highest, is still under debate. To elucidate the conflicting results, we investigated the isoform distribution in human first trimester and term placentas in situ and in vitro. Results obtained by applying immunohistochemistry, western blot, enzyme-linked immunosorbent assay (ELISA) and RT-PCR show that (1) all of the alpha1 domain-containing HLA-G isoforms are restrictedly expressed in the extravillous cytotrophoblasts (EVCTs) and very few first-trimester syncytiotrophoblasts, which directly cover cell columns, whereas mesenchymal cells of the villous chorion do not express HLA-G; (2) as demonstrated in western blots, trophoblasts express only the HLA-G1 isoform; (3) HLA-G5 and -G6 transcripts could be detected in human term placenta and isolated first-trimester trophoblasts but levels are extremely low; and (4) conditioned media of primary first-trimester trophoblasts, and the chorion laeve-derived trophoblastic cell line AC1-M59 do contain HLA-G1 fragments shed from the cell surface. Our data provide substantial evidence that none of the intron 4-containing isoforms, the so-called actively secreted, soluble HLA-G5 or -G6, are produced by human trophoblasts in situ or in vitro.

[Polytrauma with tension pneumothorax with inserted chest tube]. / Polytrauma mit Spannungspneumothorax bei liegender Thoraxdrainage.

The authors report a case of a 25-year-old woman with a polytrauma, caused by a free fall of 12 metres in suicidal intention. Following endotracheal intubation and mechanical ventilation by an emergency physician at the scene, the patient was delivered to the emergency room of an university hospital. An ultrasonic check of the abdomen revealed free fluid in the abdominal cavity, and a rupture of liver and spleen was suspected. Since breath sounds over the right lung were diminished, a chest tube was inserted immediately in the fifth intercostal space in the anterior axillary line. About 300 millilitres of blood were drained by the tube. Shortly thereafter, a laparotomy was performed, where spleen and liver rupture were confirmed and treated. After 60 minutes, the patient developed severe hypotension coupled with ventricular tachycardia and fibrillation, and resuscitation measures had to be initiated. Since breath sounds over the right lung were missing, a tension pneumothorax was suspected and a thoracotomy performed immediately. While huge amounts of air and blood were emerging from the thoracic cavity, a rupture of the right mainstem bronchus as well as of the right pulmonary artery and vena subclavia was identified. The chest tube was found dislocated into the subcutaneous tissue. Despite of open heart compression, application of adrenaline and noradrenaline and substitution of packed red blood cells and of crystalloid and colloid solutions, all resuscitation measures failed so that the patient died shortly after on the operation table. This case illustrates first the difficulties of an adequate thoracic trauma management, particularly, when clinical symptoms are discrete, second the problems of the insertion and control of a chest tube, and third risks associated with wrong position or secondary dislocation which may include - as in our case - "masking" of severe injury patterns and delay of life-saving measures such as an immediate thoracotomy. In order to improve prognosis of patients with poly-/thoracic trauma, establishment of spiral-CT in emergency centres, routine bronchoscopy and safe handling of chest tubes may be helpful.

Delays to treatment and out-of-pocket medical expenditure for tuberculosis patients, in an urban area of South America.

Short delays to treatment are important for the control of tuberculosis (TB). National Tuberculosis Programmes provide free diagnosis and treatment for smear-positive patients, so that the patients' out-of-pocket medical expenditure could be almost nil. The factors associated with delays in starting treatment, and the pre-treatment out-of-pocket medical expenditure for TB patients, have now been investigated in the Bolivian city of Cochabamba. Bolivia is the Latin American country with the highest incidence of TB. It is covered by a national TB programme that provides free diagnosis and free treatment for smear-positive patients. Structured interviews with 144 smear-positive patients enrolled in this programme revealed median patient, provider and total delays of 3.6, 6.2 and 12.9 weeks, respectively. The total delays were longer for the female patients than for the male, and for patients who consulted private doctors than for the other patients. When the first healthcare provider was a doctor, the median provider delay was 4.9 weeks in the public sector but 7.2 weeks in the private. The median out-of-pocket medical expenditure per patient, which was U.S.$13.2 overall, was much higher for those who consulted a private doctor than for those who did not (U.S.$21.9 v. U.S.$5.4, respectively; P<0.001). It appears that interventions targeting doctors (in both the private and public sectors) are likely to have a larger impact on the shortening of delays in TB treatment than interventions targeting patients. They could also reduce unnecessary out-of-pocket expenditure.

Tuberculosis control and the private health sector in Bolivia: a survey of pharmacies.

BACKGROUND: Bolivia is a high tuberculosis (TB) incidence country with a large private for-profit health sector. TB drug sales in private pharmacies are not illegal. OBJECTIVES: To measure the availability of TB drugs in private pharmacies, study vendors' attitudes, and explore the potential for collaboration between the public health sector and pharmacies. METHODS: Simulated clients visited a random sample of 100 pharmacies in the city of Cochabamba, presenting with a prescription for four TB drugs. After the survey, contacts were made with the local Pharmacist's Association. RESULTS: Twenty-five pharmacies sold at least one drug, 23 sold rifampicin and 16 sold isoniazid. Of 99 pharmacies unable to fill the whole prescription, 59 referred the client to another pharmacy, and 22 to the public services. Pharmacists said that rifampicin was often prescribed for non-TB indications, and that TB drug sales were of minimal contribution to their income. They agreed to stop selling the drugs and to refer clients seeking them to the public sector. CONCLUSION: This study has documented a small market for TB drugs sales in private pharmacies and provided the opportunity to start collaboration with the pharmacists. Our results suggest that the private sector contributes little to managing TB in Bolivia.

Nucleation and growth of molecular organic crystals in a liquid film under vapor deposition.

We report on the nucleation and growth of tetracene in a thin liquid film which is continuously supersaturated by vapor deposition of molecules onto the film. In a first stage, nucleation and fast anisotropic two-dimensional dendritic growth occurs. In a second stage, the dendrites coarsen into pallet-shaped crystals. These are highly oriented with respect to the plane of the liquid film and reach a lateral size of several 100 microm. The two-dimensional growth mode is explained by the confined growth geometry in the liquid in combination with the anisotropy of the crystal structure.