Essential role of choline for pneumococcal virulence in an experimental model of meningitis

Objectives: The goal of the present study was to elucidate the contribution of the newly recognized virulence factor choline to the pathogenesis of Streptococcus pneumoniae in an animal model of meningitis. Results: The choline containing strain D39Cho(-) and its isogenic choline-free derivative D39Cho(-)licA64 -each expressing the capsule polysaccharide 2 - were introduced intracisternally at an inoculum size of 10(3) CFU into 11 days old Wistar rats. During the first 8 h post infection both strains multiplied and stimulated a similar immune response that involved expression of high levels of proinflammatory cytokines, the matrix metalloproteinase 9 (MMP-9), IL-10, and the influx of white blood cells into the CSF. Virtually identical immune response was also elicited by intracisternal inoculation of 10(7) CFU equivalents of either choline-containing or choline-free cell walls. At sampling times past 8 h strain D39Cho(-) continued to replicate accompanied by an intense inflammatory response and strong granulocytic pleiocytosis. Animals infected with D39Cho(-) died within 20 h and histopathology revealed brain damage in the cerebral cortex and hippocampus. In contrast, the initial immune response generated by the choline-free strain D39Cho(-)licA64 began to decline after the first 8 h accompanied by elimination of the bacteria from the CSF in parallel with a strong WBC response peaking at 8 h after infection. All animals survived and there was no evidence for brain damage. Conclusion: Choline in the cell wall is essential for pneumococci to remain highly virulent and survive within the host and establish pneumococcal meningitis.

Experimental model to evaluate in vivo and in vitro cartilage MR imaging by means of histological analyses

OBJECTIVES: Implementation of an experimental model to compare cartilage MR imaging by means of histological analyses. MATERIAL AND METHODS: MRI was obtained from 4 patients expecting total knee replacement at 1.5 and/or 3T prior surgery. The timeframe between pre-op MRI and knee replacement was within two days. Resected cartilage-bone samples were tagged with Ethi((R))-pins to reproduce the histological cutting course. Pre-operative scanning at 1.5T included following parameters for fast low angle shot (FLASH: TR/TE/FA=33ms/6ms/30 degrees , BW=110kHz, 120mmx120mm FOV, 256x256 matrix, 0.65mm slice-thickness) and double echo steady state (DESS: TR/TE/FA=23.7ms/6.9ms/40 degrees , BW=130kHz, 120x120mm FOV, 256x256 matrix, 0.65mm slice-thickness). At 3T, scan parameters were: FLASH (TR/TE/FA=12.2ms/5.1ms/10 degrees , BW=130kHz, 170x170mm FOV, 320x320, 0.5mm slice-thickness) and DESS (TR/TE/FA=15.6ms/4.5ms/25 degrees , BW=200kHz, 135mmx150mm FOV, 288x320matrix, 0.5mm slice-thickness). Imaging of the specimens was done the same day at 1.5T. MRI (Noyes) and histological (Mankin) score scales were correlated using the paired t-test. Sensitivity and specificity for the detection of different grades of cartilage degeneration were assessed. Inter-reader and intra-reader reliability was determined using Kappa analysis. RESULTS: Low correlation (sensitivity, specificity) was found for both sequences in normal to mild Mankin grades. Only moderate to severe changes were diagnosed with higher significance and specificity. The use of higher field-strengths was advantageous for both protocols with sensitivity values ranging from 13.6% to 93.3% (FLASH) and 20.5% to 96.2% (DESS). Kappa values ranged from 0.488 to 0.944. CONCLUSIONS: Correlating MR images with continuous histological slices was feasible by using three-dimensional imaging, multi-planar-reformat and marker pins. The capability of diagnosing early cartilage changes with high accuracy could not be proven for both FLASH and DESS.

Inhibition of mTOR in combination with doxorubicin in an experimental model of hepatocellular carcinoma

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is resistant to chemotherapy. We reported that sirolimus, an mTOR inhibitor, has antiangiogenic properties in HCC. Since antiangiogenic therapy may enhance chemotherapy effects, we tested the antitumorigenic properties of sirolimus combined with doxorubicin in experimental HCC. METHODS: Morris Hepatoma (MH) cells were implanted into livers of syngeneic rats. Animals were assigned to sirolimus, pegylated liposomal doxorubicin, both combined or control groups. Tumoral growth was followed by MRI. Antiangiogenic effects were assessed by CD31 immunostaining and capillary tube formation assays. Cell proliferation was monitored in vitro by thymidine incorporation. Expression of p21 and phosphorylated MAPKAP kinase-2 was quantified by immunoblotting. RESULTS: Animals treated with the combination developed smaller tumors with decreased tumor microvessel density compared to animals that received monotherapies. In vitro, inhibition of mTOR further impaired capillary formation in the presence of doxorubicin. Doxorubicin reduced endothelial cell proliferation; inhibition of mTOR accentuated this effect. Doxorubicin stimulated p21 expression and the phosphorylation of MAPKAP kinase-2 in endothelial cells. Addition of mTOR inhibitor down-regulated p21, but did not decrease MAPKAP kinase-2 phosphorylation. CONCLUSIONS: Sirolimus has additive antitumoral and antiangiogenic effects when administered with doxorubicin. These findings offer a rationale for combining mTOR inhibitors with chemotherapy in HCC treatment.

Insulin-like growth factor I improves aspects of mycophenolate mofetil-impaired anastomotic healing in an experimental model

BACKGROUND: Patients taking immunosuppressants after transplantation may require intestinal surgery. Mycophenolate mofetil (MMF) has been found to impair the healing of colonic anastomoses in rats. This study examined whether insulin-like growth factor (IGF) I prevents MMF impairment of anastomotic healing. METHODS: Sixty-three rats were divided into three groups (MMF, MMF/IGF and control). Animals underwent a sigmoid colon anastomosis with a 6/0 suture, and were killed on days 2, 4 and 6 after surgery. Investigations included bursting pressure measurement, morphometric analysis, and assessment of mucosal proliferation by 5-bromo-2'-deoxyuridine and Ki67 immunohistochemistry of the anastomoses. RESULTS: The leak rate was three of 21, one of 20 and two of 20 in the MMF, MMF/IGF-I and control groups respectively. Anastomotic bursting pressures were significantly lower in the MMF group than in the control group on days 2 and 4, but there was no significant difference by day 6. Values in the MMF/IGF-I and control groups were similar. Colonic crypt depth was significantly reduced in MMF-treated animals on days 2 and 4, but this impairment was attenuated by IGF-I on day 4. Similarly, IGF-I reduced the negative impact of MMF on mucosal proliferation on days 2 and 6. CONCLUSION: Exogenous IGF-I improves some aspects of MMF-impaired anastomotic healing.

Evaluation of a new experimental model to study bone healing after ridge expansion with simultaneous implant placement--a pilot study in minipigs.

OBJECTIVE To evaluate the suitability of a minipig model for the study of bone healing and osseointegration of dental implants following bone splitting and expansion of narrow ridges. MATERIAL AND METHODS In four minipigs, the mandibular premolars and first molars were extracted together with removal of the buccal bone plate. Three months later, ridge splitting and expansion was performed with simultaneous placement of three titanium implants per quadrant. On one side of the mandible, the expanded bone gap between the implants was filled with an alloplastic biphasic calcium phosphate (BCP) material, while the gap on the other side was left unfilled. A barrier membrane was placed in half of the quadrants. After a healing period of 6 weeks, the animals were sacrificed for histological evaluation. RESULTS In all groups, no bone fractures occurred, no implants were lost, all 24 implants were osseointegrated, and the gap created by bone splitting was filled with new bone, irrespective of whether BCP or a barrier membrane was used. Slight exposure of five implants was observed, but did not lead to implant loss. The level of the most coronal bone-to-implant contact varied without being dependent on the use of BCP or a barrier membrane. In all groups, the BCP particles were not present deep in the bone-filled gap. However, BCP particles were seen at the crestal bone margin, where they were partly integrated in the new bone. CONCLUSIONS This new minipig model holds great promise for studying experimental ridge splitting/expansion. However, efforts must be undertaken to reduce implant exposure and buccal bone resorption.

Experimental Model to estimate shading losses on PV arrays

This paper presents a simple mathematical model to estimateshadinglosses on PVarrays. The model is applied directly to power calculations, without the need to consider the whole current–voltage curve. This allows the model to be used with common yield estimation software. The model takes into account both the shaded fraction of the array area and the number of blocks (a group of solar cells protected by a bypass diode) affected by shade. The results of an experimental testing campaign on several shaded PVarrays to check the validity of model are also reported.

Experimental model to estimate shading losses on PV arrays

This paper presents a simple mathematical model to estimate shading losses on PV arrays. The model is applied directly to power calculations, without the need to consider the whole current–voltage curve. This allows the model to be used with common yield estimation software. The model takes into account both the shaded fraction of the array area and the number of blocks (a group of solar cells protected by a bypass diode) affected by shade. The results of an experimental testing campaign on several shaded PV arrays to check the validity of model are also reported.

Iron chelates bind nitric oxide and decrease mortality in an experimental model of septic shock.

The hydroxamic acid siderophore ferrioxamine B [FeIII(HDFB)+] and the iron complex of diethylenetri-aminepentaacetic acid [FeIII(DTPA)2-] protected mice against death by septic shock induced by Corynebacterium parvum + lipopolysaccharide. Although FeIII(DTPA)2- was somewhat more effective than FeIII(HDFB)+, the iron-free ligand H4DFB+ was significantly more effective than DTPA. The hydroxamic acid chelator has a much higher iron affinity than the amine carboxylate, allowing for more efficient formation of the FeIII(HDFB)+ complex upon administration of the iron-free ligand. Electrochemical studies show that FeIII(DTPA)2- binds NO stoichiometrically upon reduction to iron(II) at biologically relevant potentials to form a stable NO adduct. In contrast, FeIII(HDFB)+ is a stable and efficient electrocatalyst for the reduction of NO to N2O at biologically relevant potentials. These results suggest that the mechanism of protection against death by septic shock involves NO scavenging and that particularly effective drugs that operate a low dosages may be designed based on the principle of redox catalysis. These complexes constitute a new family of drugs that rely on the special ability of transition metals to activate small molecules. In addition, the wealth of information available on siderophore chemistry and biology provides an intellectual platform for further development.

Study of salinity gradients in the Persian Gulf and an experimental model for electric energy extraction from them using nano-membranes

Salinity gradient power (SGP) is the energy that can be obtained from the mixing entropy of two solutions with a different salt concentration. River estuary, as a place for mixing salt water and fresh water, has a huge potential of this renewable energy. In this study, this potential in the estuaries of rivers leading to the Persian Gulf and the factors affecting it are analysis and assessment. Since most of the full water rivers are in the Asia, this continent with the potential power of 338GW is a second major source of energy from the salinity gradient power in the world (Wetsus institute, 2009). Persian Gulf, with the proper salinity gradient in its river estuaries, has Particular importance for extraction of this energy. Considering the total river flow into the Persian Gulf, which is approximately equal to 3486 m3/s, the amount of theoretical extractable power from salinity gradient in this region is 5.2GW. Iran, with its numerous rivers along the coast of the Persian Gulf, has a great share of this energy source. For example, with study calculations done on data from three hydrometery stations located on the Arvand River, Khorramshahr Station with releasing 1.91M/ energy which is obtained by combining 1.26m3 river water with 0.74 m3 sea water, is devoted to itself extracting the maximum amount of extractable energy. Considering the average of annual discharge of Arvand River in Khorramshahr hydrometery station, the amount of theoretical extractable power is 955 MW. Another part of parameters that are studied in this research, are the intrusion length of salt water and its flushing time in the estuary that have a significant influence on the salinity gradient power. According to the calculation done in conditions HWS and the average discharge of rivers, the maximum of salinity intrusion length in to the estuary of the river by 41km is related to Arvand River and the lowest with 8km is for Helle River. Also the highest rate of salt water flushing time in the estuary with 9.8 days is related to the Arvand River and the lowest with 3.3 days is for Helle River. Influence of these two parameters on reduces the amount of extractable energy from salinity gradient power as well as can be seen in the estuaries of the rivers studied. For example, at the estuary of the Arvand River in the interval 8.9 days, salinity gradient power decreases 9.2%. But another part of this research focuses on the design of a suitable system for extracting electrical energy from the salinity gradient. So far, five methods have been proposed to convert this energy to electricity that among them, reverse electro-dialysis (RED) method and pressure-retarded osmosis (PRO) method have special importance in practical terms. In theory both techniques generate the same amount of energy from given volumes of sea and river water with specified salinity; in practice the RED technique seems to be more attractive for power generation using sea water and river water. Because it is less necessity of salinity gradient to PRO method. In addition to this, in RED method, it does not need to use turbine to change energy and the electricity generation is started when two solutions are mixed. In this research, the power density and the efficiency of generated energy was assessment by designing a physical method. The physical designed model is an unicellular reverse electro-dialysis battery with nano heterogenic membrane has 20cmx20cm dimension, which produced power density 0.58 W/m2 by using river water (1 g NaCl/lit) and sea water (30 g NaCl/lit) in laboratorial condition. This value was obtained because of nano method used on the membrane of this system and suitable design of the cell which led to increase the yield of the system efficiency 11% more than non nano ones.

Experimental evidence and model explanation for cell population characteristics modification when applying sequential photodynamic therapy

We present experimental evidence of the existence of cell variability in terms of threshold light dose for Hep G2 (liver cancer cells) cultured. Using a theoretical model to describe the effects caused by successive photodynamic therapy (PDT) sessions, and based on the consequences of a partial response we introduce the threshold dose distribution concept within a tumor. The experimental model consists in a stack of flasks, and simulates subsequent layers of a tissue exposed to PDT application. The result indicates that cells from the same culture could respond in different ways to similar PDT induced-damages. Moreover, the consequence is a partial killing of the cells submitted to PDT, and the death fraction decreased at each in vitro PDT session. To demonstrate the occurrence of cell population modification as a response to PDT, we constructed a simple theoretical model and assumed that the threshold dose distribution for a cell population of a tumor is represented by a modified Gaussian distribution.

Mechanism and effect of esculetin in an experimental animal model of inflammatory bowel disease

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Dose-dependent effects of experimental infection with the virulent Neospora caninum Nc-Spain7 isolate in a pregnant mouse model.

Pregnant BALB/c mice have been widely used as an in vivo model to study Neospora caninum infection biology and to provide proof-of-concept for assessments of drugs and vaccines against neosporosis. The fact that this model has been used with different isolates of variable virulence, varying infection routes and differing methods to prepare the parasites for infection, has rendered the comparison of results from different laboratories impossible. In most studies, mice were infected with similar number of parasites (2 × 10(6)) as employed in ruminant models (10(7) for cows and 10(6) for sheep), which seems inappropriate considering the enormous differences in the weight of these species. Thus, for achieving meaningful results in vaccination and drug efficacy experiments, a refinement and standardization of this experimental model is necessary. Thus, 2 × 10(6), 10(5), 10(4), 10(3) and 10(2) tachyzoites of the highly virulent and well-characterised Nc-Spain7 isolate were subcutaneously inoculated into mice at day 7 of pregnancy, and clinical outcome, vertical transmission, parasite burden and antibody responses were compared. Dams from all infected groups presented nervous signs and the percentage of surviving pups at day 30 postpartum was surprisingly low (24%) in mice infected with only 10(2) tachyzoites. Importantly, infection with 10(5) tachyzoites resulted in antibody levels, cerebral parasite burden in dams and 100% mortality rate in pups, which was identical to infection with 2 × 10(6) tachyzoites. Considering these results, it is reasonable to lower the challenge dose to 10(5) tachyzoites in further experiments when assessing drugs or vaccine candidates.

A new model to study healing of a complex femur fracture with concurrent soft tissue injury in sheep

High energy bone fractures resulting from impact trauma are often accompanied by subcutaneous soft tissue injuries, even if the skin remains intact. There is evidence that such closed soft tissue injuries affect the healing of bone fractures, and vice versa. Despite this knowledge, most impact trauma studies in animals have focussed on bone fractures or soft tissue trauma in isolation. However, given the simultaneous impact on both tissues a better understanding of the interaction between these two injuries is necessary to optimise clinical treatment. The aim of this study was therefore to develop a new experimental model and characterise, for the first time, the healing of a complex fracture with concurrent closed soft tissue trauma in sheep. A pendulum impact device was designed to deliver a defined and standardised impact to the distal thigh of sheep, causing a reproducible contusion injury to the subcutaneous soft tissues. In a subsequent procedure, a reproducible femoral butterfly fracture (AO C3-type) was created at the sheep’s femur, which was initially stabilised for 5 days by an external fixator construct to allow for soft tissue swelling to recede, and ultimately in a bridging construct using locking plates. The combined injuries were applied to twelve sheep and the healing observed for four or eight weeks (six animals per group) until sacrifice. The pendulum impact led to a moderate to severe circumferential soft tissue injury with significant bruising, haematomas and partial muscle disruptions. Posttraumatic measurements showed elevated intra-compartmental pressure and circulatory tissue breakdown markers, with recovery to normal, pre-injury values within four days. Clinically, no neurovascular deficiencies were observed. Bi-weekly radiological analysis of the healing fractures showed progressive callus healing over time, with the average number of callus bridges increasing from 0.4 at two weeks to 4.2 at eight weeks. Biomechanical testing after sacrifice showed increasing torsional stiffness between four and eight weeks healing time from 10% to 100%, and increasing ultimate torsional strength from 10% to 64% (relative to the contralateral control limb). Our results demonstrate the robust healing of a complex femur fracture in the presence of a severe soft tissue contusion injury in sheep and demonstrate the establishment of a clinically relevant experimental model, for research aimed at improving the treatment of bone fractures accompanied by closed soft tissue injuries.

Effects of hyperbaric oxygen and inducible nitric oxide synthase inhibitor treatment on femoral head osteonecrosis in a rat model

Introduction: Apoptosis is the final destiny of many cells in the body, though this process has been observed in some pathological processes. One of these pathological processes is femoral head non-traumatic osteonecrosis. Among many pro/anti-apoptotic factors, nitric oxide has recently been an area of further interest. Osteocyte apoptosis and its relation to pro-apoptotic action invite further research, and the inducible form of nitric oxide synthase (iNOS)—which produces a high concentration of nitric oxide—has been flagged. The aim of this study was to investigate the effect of hyperbaric oxygen (HBO) and inducible NOS suppressor (Aminoguanidine) in prevention of femoral head osteonecrosis in an experimental model of osteonecrosis in spontaneous hypertensive rats (SHRs). Methods: After animal ethic approval 34 SHR rats were divided into four groups. Ten rats were allocated to the control group without any treatment, and eight rats were allocated to three treatment groups namely: HBO, Aminoguanidine (AMG), and the combination of HBO and AMG treatments (HBO+AMG). The HBO group received 250 kPa of oxygen via hyperbaric chamber for 30 days started at their 5th week of life; the AMG group received 1mg/ml of AMG in drinking water from the fifth week till the 17th week of life; and the last group received a combination of these treatments. Rats were sacrificed at the end of the 17th week of life and both femurs were analysed for evidence of osteonecrosis using Micro CT scan and H&E staining. Also, osteocyte apoptosis and the presence of two different forms of NOS (inducible (iNOS) and endothelial (eNOS)) were analysed by immunostaining and apoptosis staining (Hoechst and TUNEL). Results: Bone morphology of metaphyseal and epiphyseal area of all rats were investigated and analysed. Micro CT findings revealed significantly higher mean fractional trabecular bone volume (FBV) of metaphyseal area in untreated SHRs compared with all other treatments (HBO, P<0.05, HBO+AMG, P<0.005, and AMG P<0.001). Bone surface to volume ratio also significantly increased with HBO+AMG and AMG treatments when compared with the control group (18.7 Vs 20.8, P<0.05, and 18.7 Vs 21.1, P<0.05). Epiphyseal mean FBV did not change significantly among groups. In the metaphyseal area, trabecular thickness and numbers significantly decreased with AMG treatment, while trabecular separation significantly increased with both AMG and HBO+AMG treatment. Histological ratio of no ossification and osteonecrosis was 37.5%, 43.7%, 18.7% and 6.2% of control, HBO, HBO+AMG and AMG groups respectively with only significant difference observed between HBO and AMG treatment (P<0.01). High concentration of iNOS was observed in the region of osteonecrosis while there was no evidence of eNOS activity around that region. In comparison with the control group, the ratio of osteocyte apoptosis significantly reduced in AMG treatment (P<0.005). We also observed significantly fewer apoptotic osteocytes in AMG group comparing with HBO treatment (P<0.05). Conclusion: None of our treatments prevents osteonecrosis at the histological or micro CT scan level. High concentration of iNOS in the region of osteonecrosis and significant reduction of osteocyte apoptosis with AMG treatment were supportive of iNOS modulating osteocyte apoptosis in SHRs.