An efficient, non-viral dendritic vector for gene delivery in tissue engineering.

Recent developments within the field of tissue engineering (TE) have shown that biomaterial scaffold systems can be augmented via the incorporation of gene therapeutics. The objective of this study was to assess the potential of the activated polyamidoamine dendrimer (dPAMAM) transfection reagent (SuperfectTM) as a gene delivery system to mesenchymal stem cells (MSCs) in both monolayer and 3D culture on collagen based scaffolds. dPAMAM-pDNA polyplexes at a mass ratio (M:R) 10:1 (dPAMAM : pDNA) (1 ug pDNA) were capable of facilitating prolonged reporter gene expression in monolayer MSCs which was superior to that facilitated using polyethylenimine (PEI)-pDNA polyplexes (2 ug pDNA). When dPAMAM-pDNA polyplexes (1 ug pDNA) were soak loaded onto a collagen-chondroitin sulphate (CS) scaffold prolonged transgene expression was facilitated which was higher than that obtained for a PEI-pDNA polyplex (2 ug pDNA) loaded scaffold. Transgene expression was dependent on the composite nature of the collagen scaffold with varying expression profiles obtained from a suite of collagen constructs including a collagen alone, collagen-CS, collagen-hydroxyapatite, collagen-nanohydroxyapatite and collagen-hyaluronic acid scaffold. Therefore, the dPAMAM vector described herein represents a biocompatible, effective gene delivery vector for TE applications which, via matching with a particular composite scaffold type, can be tailored for regeneration of various tissue defects.

The use of existing environmental networks for the post-market monitoring of GM crop cultivation in the EU.

The European Union (EU) Directive 2001/18/EC on the deliberate release of genetically modified organisms (GMOs) into the environment requires that both Case-Specific Monitoring (CSM) and General Surveillance (GS) are considered as post-market implementing measures. Whereas CSM is directed to monitor potential adverse effects of GMOs or their use identified in the environmental risk assessment, GS aims to detect un-intended adverse effects of GMOs or their use on human and animal health or the environment. Guidance documents on the monitoring of genetically modified (GM) plants from the Commission and EFSA clarify that, as appropriate, GS can make use of established routine surveillance practices. Networks involved in routine surveillance offer recognised expertise in a particular domain and are designed to collect information on important environmental aspects over a large geographical area. However, as the suitability of existing monitoring networks to provide relevant data for monitoring impacts of GMOs is not known, plant biotechnology companies developed an approach to describe the processes and criteria that will be used for selecting and evaluating existing monitoring systems. In this paper, the availability of existing monitoring networks for this purpose is evaluated. By cataloguing the existing environmental monitoring networks in the EU, it can be concluded that they can only be used, in the context of GMO cultivation monitoring, as secondary tools to collect baseline information.

Interactions between the jasmonic and salicylic acid pathway modulate the plant metabolome and affect herbivores of different feeding types.

The phytohormones jasmonic acid (JA) and salicylic acid (SA) mediate induced plant defences and the corresponding pathways interact in a complex manner as has been shown on the transcript and proteine level. Downstream, metabolic changes are important for plant-herbivore interactions. This study investigated metabolic changes in leaf tissue and phloem exudates of Plantago lanceolata after single and combined JA and SA applications as well as consequences on chewing-biting (Heliothis virescens) and piercing-sucking (Myzus persicae) herbivores. Targeted metabolite profiling and untargeted metabolic fingerprinting uncovered different categories of plant metabolites, which were influenced in a specific manner, indicating points of divergence, convergence, positive crosstalk and pronounced mutual antagonism between the signaling pathways. Phytohormone-specific decreases of primary metabolite pool sizes in the phloem exudates may indicate shifts in sink-source relations, resource allocation, nutrient uptake or photosynthesis. Survival of both herbivore species was significantly reduced by JA and SA treatments. However, the combined application of JA and SA attenuated the negative effects at least against H. virescens suggesting that mutual antagonism between the JA and SA pathway may be responsible. Pathway interactions provide a great regulatory potential for the plant that allows triggering of appropriate defences when attacked by different antagonist species.

Familiality of neural preparation and response control in childhood attention deficit-hyperactivity disorder.

BACKGROUND: Patients with attention deficit-hyperactivity disorder (ADHD) exhibit difficulties in multiple attentional functions. Although high heritability rates suggest a strong genetic impact, aetiological pathways from genes and environmental factors to the ADHD phenotype are not well understood. Tracking the time course of deviant task processing using event-related electrophysiological brain activity should characterize the impact of familiality on the sequence of cognitive functions from preparation to response control in ADHD. Method Preparation and response control were assessed using behavioural and electrophysiological parameters of two versions of a cued continuous performance test with varying attentional load in boys with ADHD combined type (n = 97), their non-affected siblings (n = 27) and control children without a family history of ADHD (n = 43). RESULTS: Children with ADHD and non-affected siblings showed more variable performance and made more omission errors than controls. The preparatory Cue-P3 and contingent negative variation (CNV) following cues were reduced in both ADHD children and their non-affected siblings compared with controls. The NoGo-P3 was diminished in ADHD compared with controls whilst non-affected siblings were located intermediate but did not differ from both other groups. No clear familiality effects were found for the Go-P3. Better task performance was further associated with higher CNV and P3 amplitudes. CONCLUSIONS: Impairments in performance and electrophysiological parameters reflecting preparatory processes and to some extend also for inhibitory response control, especially under high attentional load, appeared to be familially driven in ADHD and may thus constitute functionally relevant endophenotypes for the disorder.

Influence of seminal plasma on fresh and post-thaw parameters of stallion epididymal spermatozoa.

Fresh and post-thaw parameters (motility, morphology and viability) of stallion epididymal spermatozoa that have been and have not been exposed to seminal plasma were evaluated, and directly compared to fresh and post-thaw parameters of ejaculated spermatozoa. Six sperm categories of each stallion (n=4) were evaluated for motility, morphology and viability. These categories were fresh ejaculated spermatozoa (Fr-E), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+), fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-), frozen-thawed ejaculated spermatozoa (Cr-E), frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma prior to freezing (Cr-SP+) and frozen-thawed epididymal spermatozoa that had never been exposed to seminal plasma (Cr-SP-). Results show that seminal plasma stimulates initial motility of fresh epididymal stallion spermatozoa while this difference in progressive motility is no longer present post-thaw; and that progressive motility of fresh or frozen-thawed ejaculated stallion spermatozoa is not always a good indicator for post-thaw progressive motility of epididymal spermatozoa. This study shows that seminal plasma has a positive influence on the incidence of overall sperm defects, midpiece reflexes and distal cytoplasmic droplets in frozen-thawed stallion epididymal spermatozoa while the occurance of midpiece reflexes is likely to be linked to distal cytoplasmic droplets. Furthermore, seminal plasma does not have an influence on viability of fresh and frozen-thawed morphologically normal epididymal spermatozoa. We recommend the retrograde flushing technique using seminal plasma as flushing medium to harvest and freeze stallion epididymal spermatozoa.

Influence of seminal plasma on fertility of fresh and frozen-thawed stallion epididymal spermatozoa.

The use of epididymal stallion spermatozoa for routine artificial insemination can secure easy future use of valuable genetics after unforeseen death or injury of a valuable stallion. The aims of this study were to (1) directly compare pregnancy rates for fresh and frozen-thawed stallion epididymal and ejaculated spermatozoa after conventional artificial insemination and (2) to investigate the effect of seminal plasma on the fertility of epididymal spermatozoa after insemination. Twenty-one mares were randomly assigned to three stallions. Mares were inseminated at five consecutive oestrous periods using fresh ejaculated spermatozoa (Fr-E, n=18), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+, n=12) or fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-, n=9), frozen-thawed ejaculated spermatozoa (Cr-E, n=18), frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma prior to freezing (Cr-SP+, n=18) and frozen-thawed epididymal spermatozoa that had never been exposed to seminal plasma (Cr-SP-, n=15). Pregnancy examinations were performed 14 days after each ovulation. Pregnancy rates were 55.6% (Fr-E, 10/18), 75% (Fr-SP+, 9/12), 22.2% (Fr-SP-, 2/9), 38.9% (Cr-E, 7/18), 27.8% (Cr-SP+, 5/18) and 6.7% (Cr-SP-, 1/15). Overall pregnancy rates for fresh and frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma were significantly better than for epididymal spermatozoa that had never been exposed to seminal plasma (P<0.05). We conclude that the exposure of stallion epididymal spermatozoa to seminal plasma improves pregnancy rates.

Duration discrimination in the range of milliseconds and seconds in children with ADHD and their unaffected siblings.

BACKGROUND: Detecting genetic factors involved in attention deficit hyperactivity disorder (ADHD) is complicated because of their small effect sizes and complex interactions. The endophenotype approach eases this by coming closer to the relevant genes. Different aspects of temporal information processing are known to be affected in ADHD. Thus, some of these aspects could represent candidate endophenotypes for ADHD. METHOD: Fifty-four sib-pairs with at least one child with ADHD and 40 control children aged 6-18 years were recruited and asked to perform two duration discrimination tasks, one with a base duration of 50 ms on automatic timing and one with a base duration of 1000 ms on cognitively controlled timing. RESULTS: Whereas children with ADHD, but not their unaffected siblings, were impaired in discrimination of longer intervals, both groups were impaired in discriminating brief intervals. Furthermore, a significant within-family correlation was found for discrimination of brief intervals. Task performances of subjects of the control group correlated with individual levels of hyperactivity/impulsivity for discrimination of brief intervals, but not of longer intervals. CONCLUSIONS: Cognitively controlled and also automatic processes of temporal information processing are impaired in children with ADHD. Discrimination of longer intervals appears as a typical 'disease marker' whereas discrimination of brief intervals shows up as a 'vulnerability marker'. Discrimination of brief intervals was found to be familial and linked to levels of hyperactivity/impulsivity. Taken together, discrimination of brief intervals represents a candidate endophenotype of ADHD.

A high-density SNP linkage scan with 142 combined subtype ADHD sib pairs identifies linkage regions on chromosomes 9 and 16.

As part of the International Multi-centre ADHD Genetics project we completed an affected sibling pair study of 142 narrowly defined Diagnostic and Statistical Manual of Mental Disorders, fourth edition combined type attention deficit hyperactivity disorder (ADHD) proband-sibling pairs. No linkage was observed on the most established ADHD-linked genomic regions of 5p and 17p. We found suggestive linkage signals on chromosomes 9 and 16, respectively, with the highest multipoint nonparametric linkage signal on chromosome 16q23 at 99 cM (log of the odds, LOD=3.1) overlapping data published from the previous UCLA (University of California, Los Angeles) (LOD>1, approximately 95 cM) and Dutch (LOD>1, approximately 100 cM) studies. The second highest peak in this study was on chromosome 9q22 at 90 cM (LOD=2.13); both the previous UCLA and German studies also found some evidence of linkage at almost the same location (UCLA LOD=1.45 at 93 cM; German LOD=0.68 at 100 cM). The overlap of these two main peaks with previous findings suggests that loci linked to ADHD may lie within these regions. Meta-analysis or reanalysis of the raw data of all the available ADHD linkage scan data may help to clarify whether these represent true linked loci.

The analysis of 51 genes in DSM-IV combined type attention deficit hyperactivity disorder: association signals in DRD4, DAT1 and 16 other genes.

Attention deficit hyperactivity disorder (ADHD) is a common neurodevelopmental disorder, starting in early childhood and persisting into adulthood in the majority of cases. Family and twin studies have demonstrated the importance of genetic factors and candidate gene association studies have identified several loci that exert small but significant effects on ADHD. To provide further clarification of reported associations and identify novel associated genes, we examined 1,038 single-nucleotide polymorphisms (SNPs) spanning 51 candidate genes involved in the regulation of neurotransmitter pathways, particularly dopamine, norepinephrine and serotonin pathways, in addition to circadian rhythm genes. Analysis used within family tests of association in a sample of 776 DSM-IV ADHD combined type cases ascertained for the International Multi-centre ADHD Gene project. We found nominal significance with one or more SNPs in 18 genes, including the two most replicated findings in the literature: DRD4 and DAT1. Gene-wide tests, adjusted for the number of SNPs analysed in each gene, identified associations with TPH2, ARRB2, SYP, DAT1, ADRB2, HES1, MAOA and PNMT. Further studies will be needed to confirm or refute the observed associations and their generalisability to other samples.

Development of Eimeria bovis in vitro: suitability of several bovine, human and porcine endothelial cell lines, bovine fetal gastrointestinal, Madin-Darby bovine kidney (MDBK) and African green monkey kidney (VERO) cells.

Several bovine, human and porcine endothelial cell lines, bovine fetal gastrointestinal cells (BFGC), Madin-Darby bovine kidney (MDBK) and African green monkey kidney (VERO) cells were exposed in vitro to sporozoites of Eimeria bovis. Parasites invaded all cells used and changed their shape to more stumpy forms within 12 h. Sporozoites left their host cells and invaded new ones frequently within the first 12 h post-infection. Further development took place only in bovine cells, although parasites survived in the other cells for at least 3 weeks. Within the non-bovine cells, conspicuously enlarged parasitophorous vacuoles developed in VERO cells and reached a diameter of 15-20 microm. The best development to first generation schizonts with regard both to time required to mature, to schizont size and to merozoite yields was observed in BFGC, followed by bovine umbilical vein and bovine spleen lymphatic endothelial cells. MDBK cells were less suitable. The life cycle was completed (development of oocysts) only occasionally in BFGC. Results are considered under several aspects. Thus, infected VERO cells may represent a suitable tool for studying the parasitophorous vacuole, while infected endothelial cells represent a system quite narrow to the in vivo situation and should allow basic studies on parasite/host cell interactions and BFGC can be used for the mass production of E. bovis first generation merozoites.

Occurrence of acute nonlymphoblastic leukemia in two girls after treatment of recurrent, disseminated Langerhans cell histiocytosis.

The occurrence of Langerhans cell histiocytosis (LCH) and acute leukemia in one individual has rarely been observed. Despite few exceptions, two distinct patterns of association appear evident: acute lymphoblastic leukemia preceding LCH and LCH preceding acute nonlymphoblastic leukemia (ANLL). The latency of ANLL after the diagnosis of LCH is suggestive of a therapy-related process. This report describes two new cases in whom ANLL was diagnosed 7 years 8 months and 5 years 8 months after the start of initial treatment of disseminated recurrent LCH. Morphology showed blasts from FAB-type M4/M5 in the first patient, who died due to progression of leukemia. The second patient showed myelodysplastic syndrome (refractory anemia with excess of blasts in transformation; RAEB-t) and is now in remission from leukemia 3 years 11 months after allogeneic bone marrow transplantation. The review of a total of 26 patients with ANLL after LCH suggests that the disease has a poor prognosis and allogeneic BMT seems to be the treatment of choice.

Microneme antigens of Eimeria bovis recognized by two monoclonal antibodies.

Two IgG1 monoclonal antibodies (mAbs 8-23F9 and 9-21G9) were developed after immunization of mice with homogenates of Eimeria bovis first-generation merozoites. Both mAbs reacted with antigens in the apical two-thirds of the parasites and immune electron microscopy determined the micronemes as targets. When tested by immunoblotting, mAb 8-23F9 failed to react with antigens separated under reducing conditions; under nonreducing conditions it recognized two components of >200 kDa. mAb 9-21G9 bound to antigens of 135 and 180 kDa after electrophoresis under reducing conditions and to a series of components when separated without reduction. The epitope of mAb 8-23F9 was destroyed by treatment of the antigen with endoglycosidase H and removal of phosphocholine (PC) by phospholipase C. Since mAb 8-23F9 does not recognize cytidine-linked PC, the data suggest that PC in combination with N-linked sugars and/or N-glycans is part of its epitope. In the case of mAb 9-21G9, endoglycosidase H did not alter the epitope. When E. bovis merozoite antigen was treated with phospholipase C the number of mAb 9-21G9-reactive constituents increased, suggesting that PC may otherwise mask the epitope. mAb 8-23F9 also bound to the apical area and the surface of E. bovis sporozoites and recognized a >200-kDa sporozoite component. When sporozoites invaded Vero cells in vitro, epitope-bearing components were released onto the host cell surface and became part of the early parasitophorous vacuole wall. At day 5 the binding of the mAb was again confined to the intracellular parasite. mAb 9-21G9 did not react with sporozoites but recognized the apical area of intra-cellular trophozoites on day 5 after invasion of host cells in vitro. When testing was done against a variety of other Apicomplexa in various assays, the only cross-reaction observed occurred with mAb 8-23F9, which bound to a conformationally determined 180-kDa component of Toxoplasma gondii cystozoites.

A monoclonal antibody reacts species-specifically with amylopectin granules of Eimeria bovis merozoites.

An IgG1 monoclonal antibody (mAb 35B9) developed against first-generation merozoites of Eimeria bovis was shown by immunoelectron microscopy to react selectively with antigens localized in amylopectin granules. Amylopectin does not contribute to the epitope, as enzymatic degradation of carbohydrates in the parasite did not alter the binding pattern of mAb 35B9. When tested by immunoblotting, despite its organelle specificity the mAb recognized a variety of E. bovis merozoite I components with predominant molecules of 135 and 200 kDa. The epitope was not affected by treatment with endoglycosidase H; thus, N-linked sugar residues should not be involved in it. Alkaline cleavage (beta-elimination), however, destroyed the epitope; thus, the involvement of O-linked carbohydrates cannot be excluded. Treatment of E. bovis merozoite extract with phospholipase C changed the binding pattern of mAb 35B9 in a way that suggests the presence of phosphorylcholine molecules on several antigens recognized by the mAb, albeit not belonging to the epitope but rather masking it. The epitope was not found in free sporozoites of E. bovis or young intracellular parasites up to day 4 after invasion of cells in vitro, whereas 5-day-old trophozoites were found to contain it. It seems to be species-specific, as it could not be shown in sporozoites or merozoites of E. tenella or in stages of several other Coccidia.

Risk of secondary leukemia after treatment with etoposide (VP-16) for Langerhans' cell histiocytosis in Italian and Austrian-German populations.

To estimate the risk of secondary leukemias after treatment with etoposide (VP-16), we evaluated subjects treated for Langerhans' cell histiocytosis (LCH) according to cooperative protocols in Italy or in Austria, Germany, Holland and Switzerland (AGDS). For each subject, information was collected on the cumulative dosages of chemotherapy and radiotherapy received, vital status and occurrence of secondary leukemia. The expected number of leukemias was estimated using age-specific incidence rates from the cancer registries in Italy and Germany. Standardized incidence ratios (SIR) were used to measure the risk of secondary leukemia among LCH patients. Five leukemias occurred among the 241 Italian study patients (SIR 520), whereas no cases were reported among the 363 AGDS patients. Interestingly, and in contrast to previous descriptions of epipodophyllotoxin-related leukemias which are mostly FAB M4 or M5, these leukemias showed typical FAB M3 features, and received a dose of VP-16 > 4,000 mg/m2. Among the AGDS cohort, very few subjects were exposed to high doses of VP-16. The risk of secondary acute non-lymphoblastic leukemia (s-ANLL) among the Italian subjects exposed to VP-16 was more than 1,000 times greater than expected. The study suggests that high doses of VP-16 appear to increase the risk of s-ANLL in LCH patients. The fact that all the leukemias described in the Italian LCH cohort were promyelocytic, and evidence of a higher incidence of promyelocytic leukemias among Italians and Latinos, suggest that high doses of etoposide in subjects of Latino origin may lead to aberrations on chromosomes 15 and 17.

Oral melanotic macules. A review of 353 cases.

Clinical and histopathologic data were obtained on 353 cases of oral melanotic macules. The mean age at the time of diagnosis for all sites was 43.1 years. A significant predilection for females (p < 0.001) was noted and the most common location was the lower lip (33.0%). The mean size of the lesions was 6.8 mm. Brown was the most common color (64.9%), and most (66.0%) of the lesions were described as flat. Melanin was evident in both the basal cell layer and lamina propria in 93.5% of the cases.

Incidence of temporomandibular joint symptoms following whiplash injury.

Recently there has been considerable litigation involving the development of temporomandibular joint (TMJ) pain and dysfunction following cervical musculoskeletal injury (whiplash). The purpose of this investigation was to interview, examine, and follow up patients with a diagnosis of whiplash injury to determine the incidence of associated temporomandibular disorders. Patients were divided into two categories: those with and those without radiologic evidence of cervical skeletal injury. In the 63 patients with radiographic evidence of cervical skeletal injury (group 1), none had clicking at the time of initial examination. In the 92 patients without positive radiographs (group 2), only one had clicking. At 1 month follow-up by telephone, 2 of 51 available patients in group 1 had developed clicking, but no new TMJ symptoms were reported by the 78 patients in group 2 contacted by phone. Seventy percent of the initial follow-up group (44 patients) with radiographic evidence of injury were contacted by telephone at 1 year and none reported new symptoms of TMJ pain or clicking. Sixty-five percent of the initial follow-up group without radiographic evidence of injury (60 patients) were interviewed and also reported no new TMJ symptoms. These data indicate that the incidence of TMJ pain and clicking following whiplash injury is extremely low, and that patients who do not have clicking on resolution of their initial pain/dysfunction subsequently do not develop this problem.

Behavioral effects of nimodipine in animals.

Neuro- and psychopharmacological effects of isopropyl-(2-methoxy-ethyl)-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate (Bay e 9736, nimodipine) are described using a variety of methods measuring behavior under normal conditions, under the influence of psychotropic drugs, as well as under the influence of ischemia or hypoxia. It has been demonstrated that nimodipine -- although not being very potent when measured in mg/kg -- exerts neuro- and psychopharmacological effects characterized by influences on the extrapyramidal system, aggressive defensive behavior, and chemically induced seizures. Electroencephalographical changes become evident whenever the normal equilibrium between cerebral catecholamine and cerebral serotonin levels is disturbed. When measured under the contingencies of a one trial passive avoidance paradigm, nimodipine is able to prevent the occurrence of retrograde amnesia in rodents after amnesiogenic events such as maximal electroconvulsive seizure or hypoxia. The substance prevents behavioral and electroencephalographic disturbances, elicited by a total cerebral ischemia, which is lethal in non-treated cats. It is concluded that nimodipine besides being a cerebrally vasoactive agent has psychopharmacological properties with a profile of actions hitherto unknown.