Influence of the developmental state of valvular lesions on the antimicrobial activity of cefotaxime in experimental enterococcal infections

Cefotaxime has little antimicrobial activity in vitro against most strains of enterococci, as measured by conventional MICs and MBCs. However, the MICs of cefotaxime against many enterococci are markedly reduced by the addition of serum to the test medium. To assess the relevance of this observation in vivo, we examined the efficacy of cefotaxime in experimental Streptococcus faecalis endocarditis. Since response to antimicrobial agents may vary with the degree of vegetation development, therapeutic efficacy was assessed both in rabbits with newly formed vegetations and in rabbits with well-developed endocardial lesions. Peak serum levels of cefotaxime (50.1 +/- 20.0 micrograms/ml) exceeded the MIC in medium supplemented with serum (4 micrograms/ml), but not in Mueller-Hinton broth alone (greater than 64 micrograms/ml). After 4 days of therapy, animals with newly formed lesions (therapy initiated 1 h after infection, transvalvular catheters removed) had lower mean vegetation bacterial titers than did untreated controls. Among animals with mature vegetations (therapy initiated 12 h after infection, catheters indwelling), the rate of mortality was significantly reduced by cefotaxime therapy. However, no difference in vegetation titers was observed. Thus, cefotaxime demonstrated antienterococcal activity within newly formed vegetations, but did not inhibit bacterial proliferation within well-established vegetations.

Growth of lung allografts after experimental transplantation

Heart and lung transplantation has been performed in cases of end-stage cardiopulmonary disease in infants. Nevertheless, it still remains unclear whether lung allografts adjust to a growing organism. In 6 young domestic pigs unilateral left lung allotransplantation was performed. Immunosuppression consisted of a triple drug therapy including cyclosporine, azathioprine, and corticosteroids. Lung growth was studied by using bronchography, pulmonary angiography, and lung histology. After 11 weeks the transplanted animals had doubled their body weight from 24 kg to 48 kg. Non-transplanted animals in contrast doubled their weight within only 6 weeks. The growth retardation was attributed to the immunosuppressive therapy. The bronchial tree and pulmonary vasculature of lung allografts showed a similar growth potential to non-transplanted lungs in animals of equivalent body weight. In one case of recurrent severe rejection of the lung no growth was observed. Therefore it was concluded that lung allografts grow adequately according to the development of the recipient organism. Lung transplantation in children does not seem to be restricted by a limited growth potential of the graft.

Intra-abdominal pressure development after different temporary abdominal closure techniques in a porcine model

BACKGROUND: Decompressive laparotomy followed by temporary abdominal closure (TAC) is an established prophylaxis and treatment for abdominal compartment syndrome. The herein presented study aimed at the comparison of volume reserve capacity and development of intra-abdominal hypertension after forced primary abdominal closure and different TAC techniques in a porcine model. METHODS: Eight anesthesized and mechanically ventilated domestic pigs underwent a standardized midline laparotomy. A bag was placed into the abdominal cavity. Before abdominal closure, the bag was prefilled with 3,000 mL water to simulate increased intra-abdominal volume. The intra-abdominal pressure (IAP) was then increased in 2 mm Hg steps up to 30 mm Hg by adding volume (volume reserve capacity) to the intra-abdominal bag. Volume reserve capacity with the corresponding IAP were analyzed and compared for primary abdominal closure, bag silo closure, a zipper system, and vacuum-assisted closure (VAC) with different negative pressures (-50, -100, and -150 mm Hg). Hemodynamic and pulmonary parameters were monitored throughout the experiment. RESULTS: Volume reserve capacity was the highest for bag silo closure followed by the zipper system and VAC with primary abdominal closure providing the least volume reserve capacity in the whole IAP range. Of interest, VAC -50 mm Hg resulted in a lower volume reserve capacity when compared with VAC -100 and -150 mm Hg. Pulmonary and hemodynamic parameters demonstrated no significant differences between primary abdominal closure and the evaluated TAC techniques at all IAP levels. CONCLUSIONS: The present experimental in vivo study indicates that bag silo closure and zipper systems may be favorable TAC techniques after decompressive laparotomy. In contrast, the VAC techniques resulted in lower volume reserve capacity and therefore may bear an increased risk for recurrent intra-abdominal hypertension in the initial phase after decompressive laparotomy.

MET Y1253D-activating point mutation and development of distant metastasis in advanced head and neck cancers

We investigated if the MET-activating point mutation Y1253D influences clinical outcomes in patients with advanced squamous cell carcinoma of the head and neck (HNSCC). The study population consisted of 152 HNSCC patients treated by hyperfractionated radiotherapy alone or concomitant with chemotherapy between September 1994 and July 2000. Tumors were screened for the presence of the MET-activating point mutation Y1253D. Seventy-eight patients (51%) received radiotherapy alone, 74 patients (49%) underwent radiotherapy concomitant with chemotherapy. Median patient age was 54 years and median follow-up was 5.5 years. Distant metastasis-free survival, local relapse-free survival and overall survival were compared with MET Y1253D status. During follow-up, 29 (19%) patients developed distant metastasis. MET Y1253D was detected in tumors of 21 out of 152 patients (14%). Distant metastasis-free survival (P = 0.008) was associated with MET Y1253D. In a multivariate Cox regression model, adjusted for T-category, only presence of MET Y1253D was associated with decreased distant metastasis-free survival: hazard ratio = 2.5 (95% confidence interval: 1.1, 5.8). The observed association between MET Y1253D-activating point mutation and decreased distant metastasis-free survival in advanced HNSCC suggests that MET may be a potential target for specific treatment interventions.

GPI-specific phospholipase D (GPI-PLD) is expressed during mouse development and is localized to the extracellular matrix of the developing mouse skeleton

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in serum and has a well-characterized biochemistry; however, its physiological role is completely unknown. Previous investigations into GPI-PLD have focused on the adult animal or on in vitro systems and a putative role in development has been neither proposed nor investigated. We describe the first evidence of GPI-PLD expression during mouse embryonic ossification. GPI-PLD expression was detected predominantly at sites of skeletal development, increasing during the course of gestation. GPI-PLD was observed during both intramembraneous and endochondral ossification and localized predominantly to the extracellular matrix of chondrocytes and to primary trabeculae of the skeleton. In addition, the mouse chondrocyte cell line ATDC5 expressed GPI-PLD after experimental induction of differentiation. These results implicate GPI-PLD in the process of bone formation during mouse embryogenesis.

Experimental plant communities develop phylogenetically overdispersed abundance distributions during assembly

The importance of competition between similar species in driving community assembly is much debated. Recently, phylogenetic patterns in species composition have been investigated to help resolve this question: phylogenetic clustering is taken to imply environmental filtering, and phylogenetic overdispersion to indicate limiting similarity between species. We used experimental plant communities with random species compositions and initially even abundance distributions to examine the development of phylogenetic pattern in species abundance distributions. Where composition was held constant by weeding, abundance distributions became overdispersed through time, but only in communities that contained distantly related clades, some with several species (i.e., a mix of closely and distantly related species). Phylogenetic pattern in composition therefore constrained the development of overdispersed abundance distributions, and this might indicate limiting similarity between close relatives and facilitation/complementarity between distant relatives. Comparing the phylogenetic patterns in these communities with those expected from the monoculture abundances of the constituent species revealed that interspecific competition caused the phylogenetic patterns. Opening experimental communities to colonization by all species in the species pool led to convergence in phylogenetic diversity. At convergence, communities were composed of several distantly related but species-rich clades and had overdispersed abundance distributions. This suggests that limiting similarity processes determine which species dominate a community but not which species occur in a community. Crucially, as our study was carried out in experimental communities, we could rule out local evolutionary or dispersal explanations for the patterns and identify ecological processes as the driving force, underlining the advantages of studying these processes in experimental communities. Our results show that phylogenetic relations between species provide a good guide to understanding community structure and add a new perspective to the evidence that niche complementarity is critical in driving community assembly.

Influence of human saliva on the development of artificial erosions

It was hypothesized that saliva from patients with erosion exhibits lower protective efficacy compared to saliva from patients without erosion, based on in vitro enamel softening studies. A total of 645 enamel specimens were distributed among seven experimental groups. Saliva was gathered from each of 10 volunteers without clinical signs of dental erosion and from 10 patients exhibiting severe erosive defects. Aliquots of 50 ml of saliva from each patient were mixed with sour drops or citric acid, respectively. Pooled saliva, sour drops and citric acid mixed with water served as controls. The enamel specimens were soaked in the respective mixture for 5 min and were subsequently incubated in pure saliva for 2 min. This cycle was repeated three times, then the specimens were kept in 100 ml of saliva for 8 h. Surface microhardness was evaluated at the beginning of the experiment and after each cycle. During the experiments, microhardness decreased significantly in all groups except for the pure saliva group. For sour drops and citric acid mixed with saliva from patients without erosion, the final microhardness was higher compared to the mixture of the two erosive compounds with saliva from patients with erosion. The storage of saliva for 8 h resulted in a certain amount of rehardening, with the highest level of rehardening being observed in the group that was least demineralized (sour drops plus saliva from patients without erosion). It is concluded that salivary components play a crucial role in the development of dental erosion.

Development of an ultrasound-guided technique for pudendal nerve block in cat cadavers.

The objective of this prospective experimental cadaveric study was to develop an ultrasound-guided technique to perform an anaesthetic pudendal nerve block in male cats. Fifteen fresh cadavers were used for this trial. A detailed anatomical dissection was performed on one cat in order to scrutinise the pudendal nerve and its ramifications. In a second step, the cadavers of six cats were used to test three different ultrasonographic approaches to the pudendal nerve: the deep dorso-lateral, the superficial dorso-lateral and the median transperineal. Although none of the approaches allowed direct ultrasonographical identification of the pudendal nerve branches, the deep dorso-lateral was found to be the most advantageous one in terms of practicability and ability to identify useful and reliable landmarks. Based on these findings, the deep dorso-lateral approach was selected as technique of choice for tracer injections (0.1 ml 1% methylene blue injected bilaterally) in six cat cadavers distinct from those used for the ultrasonographical study. Anatomical dissection revealed a homogeneous spread of the tracer around the pudendal nerve sensory branches in all six cadavers. Finally, computed tomography was performed in two additional cadavers after injection of 0.3 ml/kg (0.15 ml/kg per each injection sites, left and right) contrast medium through the deep dorso-lateral approach in order to obtain a model of volume distribution applicable to local anaesthetics. Our findings in cat cadavers indicate that ultrasound-guided pudendal nerve block is feasible and could be proposed to provide peri-operative analgesia in clinical patients undergoing perineal urethrostomy.

T-cadherin loss promotes experimental metastasis of squamous cell carcinoma

T-cadherin is gaining recognition as a determinant for the development of incipient invasive squamous cell carcinoma (SCC). However, effects of T-cadherin expression on the metastatic potential of SCC have not been studied. Here, using a murine model of experimental metastasis following tail vein injection of A431 SCC cells we report that loss of T-cadherin increased both the incidence and rate of appearance of lung metastases. T-cadherin-silenced SCC metastases were highly disordered with evidence of single cell dissemination away from main foci whereas SCC metastases overexpressing T-cadherin developed as compact, tightly organised sheets. SCC cell adhesion to vascular endothelial cells (EC) in culture was increased for T-cadherin-silenced SCC and decreased for T-cadherin-overexpressing SCC. Confocal microscopy showed that T-cadherin-silenced SCC adherent on EC display an elongated morphology with long thin extensions and a high degree of intercalation within the EC monolayer, whereas SCC overexpressing T-cadherin formed poorly-spread multicellular aggregates that remain on the outer surface of the EC monolayer. T-cadherin-deficient SCC or human keratinocyte cells exhibited increased transendothelial migration in vitro which could be attenuated in the presence of EGFR inhibitor gefitinib. Our data suggest that loss of T-cadherin can increase metastatic potential and aggressiveness of SCC, possibly due to facilitating arrest and extravasation through the vascular wall and/or more efficient establishment of metastases in the new microenvironment.

Acquired thrombotic thrombocytopenic purpura. Development of an autoimmune response

The von Willebrand factor (VWF)-cleaving metalloprotease, ADAMTS13 (adisintegrin and metalloprotease with thrombospondin type 1 motifs-13) is the only known target of the dysregulated immune response in acquired TTP. Autoantibodies to ADAMTS13 either neutralize its activity or accelerate its clearance, thereby causing a severe deficiency of ADAMTS13 in plasma. As a consequence, size regulation of VWF is impaired and the persistence of ultra-large VWF (ULVWF) multimers facilitates microvascular platelet aggregation causing microangiopathic haemolytic anaemia and ischaemic organ damage. Autoimmune TTP although a rare disease with an annual incidence of 1.72 cases has a mortality rate of 20% even with adequate therapy. We describe the mechanisms involved in ADAMTS13 autoimmunity with a focus on the role of B- and T-cells in the pathogenesis of this disorder. We discuss the potential translation of recent experimental findings into future therapeutic concepts for the treatment of acquired TTP.

Experimental Validation of Finite Element Analysis of Human Vertebral Collapse Under Large Compressive Strains

Osteoporosis-related vertebral fractures represent a major health problem in elderly populations. Such fractures can often only be diagnosed after a substantial deformation history of the vertebral body. Therefore, it remains a challenge for clinicians to distinguish between stable and progressive potentially harmful fractures. Accordingly, novel criteria for selection of the appropriate conservative or surgical treatment are urgently needed. Computer tomography-based finite element analysis is an increasingly accepted method to predict the quasi-static vertebral strength and to follow up this small strain property longitudinally in time. A recent development in constitutive modeling allows us to simulate strain localization and densification in trabecular bone under large compressive strains without mesh dependence. The aim of this work was to validate this recently developed constitutive model of trabecular bone for the prediction of strain localization and densification in the human vertebral body subjected to large compressive deformation. A custom-made stepwise loading device mounted in a high resolution peripheral computer tomography system was used to describe the progressive collapse of 13 human vertebrae under axial compression. Continuum finite element analyses of the 13 compression tests were realized and the zones of high volumetric strain were compared with the experiments. A fair qualitative correspondence of the strain localization zone between the experiment and finite element analysis was achieved in 9 out of 13 tests and significant correlations of the volumetric strains were obtained throughout the range of applied axial compression. Interestingly, the stepwise propagating localization zones in trabecular bone converged to the buckling locations in the cortical shell. While the adopted continuum finite element approach still suffers from several limitations, these encouraging preliminary results towardsthe prediction of extended vertebral collapse may help in assessing fracture stability in future work.

Matrix metalloproteinase inhibition lowers mortality and brain injury in experimental pneumococcal meningitis

Pneumococcal meningitis (PM) results in high mortality rates and long-lasting neurological deficits. Hippocampal apoptosis and cortical necrosis are histopathological correlates of neurofunctional sequelae in rodent models and are frequently observed in autopsy studies of patients who die of PM. In experimental PM, inhibition of matrix metalloproteinases (MMPs) and/or tumor necrosis factor (TNF)-converting enzyme (TACE) has been shown to reduce brain injury and the associated impairment of neurocognitive function. However, none of the compounds evaluated in these studies entered clinical development. Here, we evaluated two second-generation MMP and TACE inhibitors with higher selectivity and improved oral availability. Ro 32-3555 (Trocade, cipemastat) preferentially inhibits collagenases (MMP-1, -8, and -13) and gelatinase B (MMP-9), while Ro 32-7315 is an efficient inhibitor of TACE. PM was induced in infant rats by the intracisternal injection of live Streptococcus pneumoniae. Ro 32-3555 and Ro 32-7315 were injected intraperitoneally, starting at 3 h postinfection. Antibiotic (ceftriaxone) therapy was initiated at 18 h postinfection, and clinical parameters (weight, clinical score, mortality rate) were recorded. Myeloperoxidase activities, concentrations of cytokines and chemokines, concentrations of MMP-2 and MMP-9, and collagen concentrations were measured in the cerebrospinal fluid. Animals were sacrificed at 42 h postinfection, and their brains were assessed by histomorphometry for hippocampal apoptosis and cortical necrosis. Both compounds, while exhibiting disparate MMP and TACE inhibitory profiles, decreased hippocampal apoptosis and cortical injury. Ro 32-3555 reduced mortality rates and cerebrospinal fluid TNF, interleukin-1β (IL-1β) and collagen levels, while Ro 32-7315 reduced weight loss and cerebrospinal fluid TNF and IL-6 levels.

Vitamin B6 reduces hippocampal apoptosis in experimental pneumococcal meningitis

BACKGROUND Bacterial meningitis caused by Streptococcus pneumoniae leads to death in up to 30% of patients and leaves up to half of the survivors with neurological sequelae. The inflammatory host reaction initiates the induction of the kynurenine pathway and contributes to hippocampal apoptosis, a form of brain damage that is associated with learning and memory deficits in experimental paradigms. Vitamin B6 is an enzymatic cofactor in the kynurenine pathway and may thus limit the accumulation of neurotoxic metabolites and preserve the cellular energy status. The aim of this study in a pneumococcal meningitis model was to investigate the effect of vitamin B6 on hippocampal apoptosis by histomorphology, by transcriptomics and by measurement of cellular nicotine amide adenine dinucleotide content. METHODS AND RESULTS Eleven day old Wistar rats were infected with 1x10(6) cfu/ml of S. pneumoniae and randomized for treatment with vitamin B6 or saline as controls. Vitamin B6 led to a significant (p > 0.02) reduction of hippocampal apoptosis. According to functional annotation based clustering, vitamin B6 led to down-regulation of genes involved in processes of inflammatory response, while genes encoding for processes related to circadian rhythm, neuronal signaling and apoptotic cell death were mostly up-regulated. CONCLUSIONS Our results provide evidence that attenuation of apoptosis by vitamin B6 is multi-factorial including down-modulation of inflammation, up-regulation of the neuroprotective brain-derived neurotrophic factor and prevention of the exhaustion of cellular energy stores. The neuroprotective effect identifies vitamin B6 as a potential target for the development of strategies to attenuate brain injury in bacterial meningitis.

Alphaviral cytotoxicity and its implication in vector development

A great variety of viruses have been engineered to serve as expression vectors. Among them, the alphaviruses Semliki Forest virus and Sindbis virus represent promising tools for heterologous gene expression in a wide variety of host cells. Several applications have already been described in neurobiological studies, in gene therapy, for vaccine development and in cancer therapy. Both viruses trigger stress pathways in the cells they infect, sometimes culminating in the death of the host. This inherent property is either an advantage or a drawback, depending on the type of application. This review covers the development and applications of alphavirus vectors and, as our work has been mainly with Semliki Forest virus, we have focused on this virus with special emphasis on how the understanding of Semliki Forest virus cytotoxicity enables it to be manipulated and used.