Tissue response and wound healing after placement of two types of bioengineered grafts containing vital cells in submucosal maxillary pouches: an experimental pilot study in rabbits

PURPOSE: This pilot study evaluated the wound healing and tissue response after placement of two different skin substitutes in subgingival mucosal pouches in rabbits. MATERIALS AND METHODS: Four rabbits were selected to receive a commercially available skin substitute consisting of a collagen matrix with fibroblasts and an epithelial layer (test membrane 1) and a prototype device consisting of a collagen matrix with fibroblasts only (test membrane 2). In each rabbit, two horizontal incisions were made in the buccal alveolar mucosa of the maxilla bilaterally to create submucosal pouches. Three pouches in each animal were filled with either the test 1 or test 2 membranes, and one pouch was left without a membrane (sham-operated control). All rabbits were sacrificed after a healing period of 4 weeks, and histologic samples were prepared and examined. RESULTS: After a healing period of 1 month, both tested membranes were still visible in the sections. Test membrane 1 was still bilayered, contained inflammatory cells in its center, and was encapsulated by a thick fibrous tissue. Numerous ectopic calcifications were evident in the collagenous part of the membrane and in association with some basal epithelial cells. Test membrane 2 was also encapsulated in fibrous tissue, with inflammatory cells present only between the fibrous encapsulation and the remnants of the membrane. For test membrane 2, no calcifications were visible. CONCLUSIONS: Test membrane 1 seemed to be more resistant to degradation, but there was also a more pronounced inflammatory reaction in comparison to test membrane 2, especially in the vicinity of the keratinocytes. The significance of the ectopic calcifications, along with that of the resorption or degradation processes of both tested membranes, must be evaluated in future experimental studies, with different time points after implantation examine

Fatty acid and peptide profiles in plasma membrane and membrane rafts of PUFA supplemented RAW264.7 macrophages

The eukaryotic cell membrane possesses numerous complex functions, which are essential for life. At this, the composition and the structure of the lipid bilayer are of particular importance. Polyunsaturated fatty acids may modulate the physical properties of biological membranes via alteration of membrane lipid composition affecting numerous physiological processes, e.g. in the immune system. In this systematic study we present fatty acid and peptide profiles of cell membrane and membrane rafts of murine macrophages that have been supplemented with saturated fatty acids as well as PUFAs from the n-3, the n-6 and the n-9 family. Using fatty acid composition analysis and mass spectrometry-based peptidome profiling we found that PUFAs from both the n-3 and the n-6 family have an impact on lipid and protein composition of plasma membrane and membrane rafts in a similar manner. In addition, we found a relation between the number of bis-allyl-methylene positions of the PUFA added and the unsaturation index of plasma membrane as well as membrane rafts of supplemented cells. With regard to the proposed significance of lipid microdomains for disease development and treatment our study will help to achieve a targeted dietary modulation of immune cell lipid bilayers.

Enhancing sealing of fetal membrane defects using tissue engineered native amniotic scaffolds in the rabbit model

OBJECTIVE: The purpose of this study was to compare the efficacy of native engineered amniotic scaffolds (AS) and polyesterurethane scaffolds (DegraPol) and document wound healing response when sealing iatrogenic fetal membrane defects in the rabbit model. STUDY DESIGN: Native AS were engineered from freshly harvested membranes of 23 days' gestational age (GA; term = 31-2 d). Acellularity of AS was assessed by histology, light and scanning electron microscopy. Fetal membrane defects were created by 14 gauge-needle puncture at GA 23 days and primarily closed with AS (n = 10) or DegraPol (n = 10) or left unclosed (positive controls; n = 10). Sixty-one sacs served as negative controls. At GA 30 days a second look hysterotomy was performed to assess presence of amniotic fluid (AF) and harvest plugging sites for microscopic evaluation. RESULTS: Engineered AS had a cell-free collagenous fiber network. AF was significantly higher only in the DegraPol group (78%; P < .05) compared to the AF in positive controls (17%). Integration of plugs in the fetal membrane defect was better with AS than DegraPol, with higher reepithelialization rates (AS: 52.5% +/- 6.5%; DegraPol: 11.6% +/- 2.6%; P < .001) and proliferation indices (AS: 0.47 +/- 0.03; DegraPol: 0.28 +/- 0.04; P = .001). In both treatment groups, cell proliferation in the myometrium was increased (P < .05). CONCLUSION: Native AS seal iatrogenic fetal membrane defects better than DegraPol. Within a week, there is abundant reepithelilization and minimal local inflammation. This yields the proof of principle that engineered native, amniotic membrane scaffolds enhance fetal membrane wound healing response.

The basophil activation test in immediate-type drug allergy

Diagnosis of drug allergy involves first the recognition of sometimes unusual symptoms as drug allergy and, second, the identification of the eliciting drug. This is an often difficult task, as the clinical picture and underlying pathomechanisms are heterogeneous. In clinical routine, physicians frequently have to rely upon a suggestive history and eventual provocation tests, both having their specific limitations. For this reason both in vivo (skin tests) and in vitro tests are investigated intensively as tools to identify the disease-eliciting drug. One of the tests evaluated in drug allergy is the basophil activation test (BAT). Basophils with their high-affinity IgE receptors are easily accessible and therefore can be used as indicator cells for IgE-mediated reactions. Upon allergen challenge and cross-linking of membrane-bound IgE antibodies (via Fc-epsilon-RI) basophils up-regulate certain activation markers on their surface such as CD63 and CD203c, as well as intracellular markers (eg, phosphorylated p38MAPK). In BAT, these alterations can be detected rapidly on a single-cell basis by multicolor flow cytometry using specific monoclonal antibodies. Combining this technique with in vitro passive sensitization of donor basophils with patients' serum, one can prove the IgE dependence of a drug reaction. This article summarizes the authors' current experience with the BAT in the diagnostic management of immediate-type drug allergy mediated by drug-specific IgE antibodies.

Ceramide in Plasma Membrane Repair

The perforation of the plasmalemma by pore-forming toxins causes an influx of Ca2+ and an efflux of cytoplasmic proteins. In order to ensure cellular survival, lesions have to be identified, plugged and removed from the membrane. The Ca2+-driven fusion of lysosomes with the plasma membrane leads to hydrolysis of sphingomyelin by acid sphingomyelinase and a formation of ceramide platforms in the outer leaflet of the lipid bilayer. We propose that the negative curvature, promoted by tighter packing of lipids in the outer layer, leads to an inward vesiculation of the damaged area for its endocytotic uptake and internal degradation. In contrast, the activation of neutral sphingomyelinase triggers the production of ceramide within the inner leaflet of the lipid bilayer, thereby promoting an outward curvature, which enables the cell to shed the membrane-containing toxin pore into the extracellular space. In this process, ceramide is supported by members of the annexin protein family which act as Ca2+ sensors and as membrane fusion agents.

High resolution immunoelectron microscopic localization of functional domains of laminin, nidogen, and heparan sulfate proteoglycan in epithelial basement membrane of mouse cornea reveals different topological orientations

Thin and ultrathin cryosections of mouse cornea were labeled with affinity-purified antibodies directed against either laminin, its central segments (domain 1), the end of its long arm (domain 3), the end of one of its short arms (domain 4), nidogen, or low density heparan sulfate proteoglycan. All basement membrane proteins are detected by indirect immunofluorescence exclusively in the epithelial basement membrane, in Descemet's membrane, and in small amorphous plaques located in the stroma. Immunoelectron microscopy using the protein A-gold technique demonstrated laminin domain 1 and nidogen in a narrow segment of the lamina densa at the junction to the lamina lucida within the epithelial basement membrane. Domain 3 shows three preferred locations at both the cellular and stromal boundaries of the epithelial basement membrane and in its center. Domain 4 is located predominantly in the lamina lucida and the adjacent half of the lamina densa. The low density heparan sulfate proteoglycan is found all across the basement membrane showing a similar uniform distribution as with antibodies against the whole laminin molecule. In Descemet's membrane an even distribution was found with all these antibodies. It is concluded that within the epithelial basement membrane the center of the laminin molecule is located near the lamina densa/lamina lucida junction and that its long arm favors three major orientations. One is close to the cell surface indicating binding to a cell receptor, while the other two are directed to internal matrix structures. The apparent codistribution of laminin domain 1 and nidogen agrees with biochemical evidence that nidogen binds to this domain.

Evidence for Emergency Vaccination Having Played a Crucial Role to Control the 1965/66 Foot-and-Mouth Disease Outbreak in Switzerland.

Foot-and-mouth disease (FMD) is a highly contagious disease that caused several large outbreaks in Europe in the last century. The last important outbreak in Switzerland took place in 1965/66 and affected more than 900 premises and more than 50,000 animals were slaughtered. Large-scale emergency vaccination of the cattle and pig population has been applied to control the epidemic. In recent years, many studies have used infectious disease models to assess the impact of different disease control measures, including models developed for diseases exotic for the specific region of interest. Often, the absence of real outbreak data makes a validation of such models impossible. This study aimed to evaluate whether a spatial, stochastic simulation model (the Davis Animal Disease Simulation model) can predict the course of a Swiss FMD epidemic based on the available historic input data on population structure, contact rates, epidemiology of the virus, and quality of the vaccine. In addition, the potential outcome of the 1965/66 FMD epidemic without application of vaccination was investigated. Comparing the model outcomes to reality, only the largest 10% of the simulated outbreaks approximated the number of animals being culled. However, the simulation model highly overestimated the number of culled premises. While the outbreak duration could not be well reproduced by the model compared to the 1965/66 epidemic, it was able to accurately estimate the size of the area infected. Without application of vaccination, the model predicted a much higher mean number of culled animals than with vaccination, demonstrating that vaccination was likely crucial in disease control for the Swiss FMD outbreak in 1965/66. The study demonstrated the feasibility to analyze historical outbreak data with modern analytical tools. However, it also confirmed that predicted epidemics from a most carefully parameterized model cannot integrate all eventualities of a real epidemic. Therefore, decision makers need to be aware that infectious disease models are useful tools to support the decision-making process but their results are not equal valuable as real observations and should always be interpreted with caution.

Excitability properties of mouse motor axons in the mutant SOD1(G93A) model of amyotrophic lateral sclerosis

Non-invasive excitability studies of motor axons in patients with amyotrophic lateral sclerosis (ALS) have revealed a changing pattern of abnormal membrane properties with disease progression, but the heterogeneity of the changes has made it difficult to relate them to pathophysiology. The SOD1(G93A) mouse model of ALS displays more synchronous motoneuron pathology. Multiple excitability measures of caudal and sciatic nerves in mutant and wild-type mice were compared before onset of signs and during disease progression (4-19 weeks), and they were related to changes in muscle fiber histochemistry. Excitability differences indicated a modest membrane depolarization in SOD1(G93A) axons at about the time of symptom onset (8 weeks), possibly due to deficient energy supply. Previously described excitability changes in ALS patients, suggesting altered sodium and potassium conductances, were not seen in the mice. This suggests that those changes relate to features of the human disease that are not well represented in the animal model.

Garlic extract induces intestinal P-glycoprotein, but exhibits no effect on intestinal and hepatic CYP3A4 in humans

Garlic extracts have been shown to decrease drug exposure for saquinavir, a P-glycoprotein and cytochrome P450 3A4 substrate. In order to explore the underlying mechanisms and to study the effects of garlic on pre-systemic drug elimination, healthy volunteers were administered garlic extract for 21 days. Prior to and at the end of this period, expression of duodenal P-glycoprotein and cytochrome P450 3A4 protein were assayed and normalized to villin, while hepatic cytochrome P450 3A4 function and simvastatin, pravastatin and saquinavir pharmacokinetics were also evaluated. Ingestion of garlic extract increased expression of duodenal P-glycoprotein to 131% (95% CI, 105-163%), without increasing the expression of cytochrome P450 3A4 which amounted to 87% (95% CI, 67-112%), relative to baseline in both cases. For the erythromycin breath test performed, the average result was 96% (95% CI, 83-112%). Ingestion of garlic extract had no effect on drug and metabolite AUCs following a single dose of simvastatin or pravastatin, although the average area under the plasma concentration curve (AUC) of saquinavir decreased to 85% (95% CI, 66-109%), and changes in intestinal P-glycoprotein expression negatively correlated with this change. In conclusion, garlic extract induces intestinal expression of P-glycoprotein independent of cytochrome P450 3A4 in human intestine and liver.

Muscle ischaemia in patients with orthostatic hypotension assessed by velocity recovery cycles

Patients with orthostatic hypotension may experience neck pain radiating to the occipital region of the skull and the shoulders while standing (so-called coat-hanger ache). This study assessed muscle membrane potential in the trapezius muscle of patients with orthostatic hypotension and healthy subjects during head-up tilt (HUT), by measuring velocity recovery cycles (VRCs) of muscle action potentials as an indicator of muscle membrane potential.

Long-term stability of surgical bone regenerative procedures of peri-implantitis lesions in a prospective case-control study over 3 years

Objectives: To evaluate the extent of bone fill over 3 years following the surgical treatment of peri-implantitis with bone grafting with or without a membrane. Material and Methods: In a non-submerged wound-healing mode, 15 subjects with 27 implants were treated with a bone substitute (Algipore®) alone and 17 subjects with 29 implants were treated with the bone substitute and a resorbable membrane (Osseoquest®). Implants with radiographic bone loss ≥1.8 mm following the first year in function and with bleeding and/or pus on probing were included. Following surgery, subjects were given systemic antibiotics (10 days) and rinsed with chlorhexidine. After initial healing, the subjects were enrolled in a strict maintenance programme. Results: Statistical analysis failed to demonstrate changes in bone fill between 1 and 3 years both between and within procedure groups. The mean defect fill at 3 years was 1.3 ± (SD) 1.3 mm if treated with the bone substitute alone and 1.6 ± (SD) 1.2 mm if treated with an adjunct resorbable membrane, (p=0.40). The plaque index decreased from approximately 40–10%, remaining stable during the following 2 years. Conclusion: Defect fill using a bone substitute with or without a membrane technique in the treatment of peri-implantitis can be maintained over 3 years.

Cancer therapy modulates VEGF signaling and viability in adult rat cardiac microvascular endothelial cells and cardiomyocytes

This work was motivated by the incomplete characterization of the role of vascular endothelial growth factor-A (VEGF-A) in the stressed heart in consideration of upcoming cancer treatment options challenging the natural VEGF balance in the myocardium. We tested, if the cytotoxic cancer therapy doxorubicin (Doxo) or the anti-angiogenic therapy sunitinib alters viability and VEGF signaling in primary cardiac microvascular endothelial cells (CMEC) and adult rat ventricular myocytes (ARVM). ARVM were isolated and cultured in serum-free medium. CMEC were isolated from the left ventricle and used in the second passage. Viability was measured by LDH-release and by MTT-assay, cellular respiration by high-resolution oxymetry. VEGF-A release was measured using a rat specific VEGF-A ELISA-kit. CMEC were characterized by marker proteins including CD31, von Willebrand factor, smooth muscle actin and desmin. Both Doxo and sunitinib led to a dose-dependent reduction of cell viability. Sunitinib treatment caused a significant reduction of complex I and II-dependent respiration in cardiomyocytes and the loss of mitochondrial membrane potential in CMEC. Endothelial cells up-regulated VEGF-A release after peroxide or Doxo treatment. Doxo induced HIF-1α stabilization and upregulation at clinically relevant concentrations of the cancer therapy. VEGF-A release was abrogated by the inhibition of the Erk1/2 or the MAPKp38 pathway. ARVM did not answer to Doxo-induced stress conditions by the release of VEGF-A as observed in CMEC. VEGF receptor 2 amounts were reduced by Doxo and by sunitinib in a dose-dependent manner in both CMEC and ARVM. In conclusion, these data suggest that cancer therapy with anthracyclines modulates VEGF-A release and its cellular receptors in CMEC and ARVM, and therefore alters paracrine signaling in the myocardium.

Attempted suicide in shanghai districts: A pilot study

This is a pilot study whose objective was to collect data on attempted suicide in 5 districts of Shanghai and to test the feasibility of introducing an ongoing monitoring of attempted suicide. Data on a total of 363 cases were collected. The mean age of the patients was 33 years, 67% being female. Ingesting drugs or other chemical substances was the main method used for self-harm. Reasons for attempted suicide in these districts of Shanghai often appear to be related to family conflicts and unemployment. In spite of methodological limitations, the recorded data allow some preliminary conclusions regarding the characteristics of patients in districts of Shanghai admitted after a suicide attempt. Continuous monitoring of attempted suicide in this urban area of China should be established and data collection improved to raise awareness in health professionals and to develop preventive measures geared toward the needs of these patients.