Predicting Patient Status in Chronic Thromboembolic Pulmonary Hypertension Using a Biophysical Model.

Chronic thromboembolic pulmonary hypertension (CTEPH) involves abnormally high blood pressure in the pulmonary vessels and is associated with small vessel vasculopathy and pre-capillary proximal occlusions. Management of CTEPH disease is challenging, therefore accurate diagnosis is crucial in ensuring effective treatment and improved patient outcomes. The treatment of choice for CTEPH is pulmonary endarterectomy, which is an invasive surgical intervention to remove thrombi. Following PEA, a number of patients experience poor outcomes or worse-than-expected improvements, which may indicate that they have significant small vessel disease. A method that can predict the extent of distal remodelling may provide useful clinical information to plan appropriate CTEPH patient treatment. Here, a novel biophysical modelling approach has been developed to estimate and quantify the extent of distal remodelling. This method includes a combination of mathematical modelling and computed tomography pulmonary angiography to first model the geometry of the pulmonary arteries and to identify the under-perfused regions in CTEPH. The geometric model is then used alongside haemodynamic measurements from right heart catheterisation to predict distal remodelling. In this study, the method is tested and validated using synthetically generated remodelling data. Then, a preliminary application of this technique to patient data is shown to demonstrate the potential of the approach for use in the clinical setting.Clinical relevance- Patient-specific modelling can help provide useful information regarding the extent of distal vasculopathy on a per-patient basis, which remains challenging. Physicians can be unsure of outcomes following pulmonary endarterectomy. Therefore, the predictive aspect of the patient's response to surgery can help with clinical decision-making.

An in silico approach to understanding the interaction between cardiovascular and pulmonary lymphatic dysfunction.

The lung is extremely sensitive to interstitial fluid balance, yet the role of pulmonary lymphatics in lung fluid homeostasis and its interaction with cardiovascular pressures is poorly understood. In health, there is a fine balance between fluid extravasated from the pulmonary capillaries into the interstitium and the return of fluid to the circulation via the lymphatic vessels. This balance is maintained by an extremely interdependent system governed by pressures in the fluids (air and blood) and tissue (interstitium), lung motion during breathing, and the permeability of the tissues. Chronic elevation in left atrial pressure (LAP) due to left heart disease increases the capillary blood pressure. The consequent fluid accumulation in the delicate lung tissue increases its weight, decreases its compliance, and impairs gas exchange. This interdependent system is difficult, if not impossible, to study experimentally. Computational modeling provides a unique perspective to analyze fluid movement in the cardiopulmonary vasculature in health and disease. We have developed an initial in silico model of pulmonary lymphatic function using an anatomically structured model to represent ventilation and perfusion and underlying biophysical laws governing fluid transfer at the interstitium. This novel model was tested against increased LAP and noncardiogenic effects (increased permeability). The model returned physiologically reasonable values for all applications, predicting pulmonary edema when LAP reached 25 mmHg and with increased permeability.NEW & NOTEWORTHY This model presents a novel approach to understanding the interaction between cardiac dysfunction and pulmonary lymphatic function, using anatomically structured models and biophysical equations to estimate regional variation in fluid transport from blood to interstitial and lymphatic flux. This fluid transport model brings together advanced models of ventilation, perfusion, and lung mechanics to produce a detailed model of fluid transport in health and various altered pathological conditions.

Tissue Engineered Human Amniotic Membrane Application in Mouse Ovarian Follicular Culture.

Since folliculogenesis requires a powerful cell-matrix interaction, natural scaffolds seem to be needed for follicular culture. Human amniotic membrane (HAM) offers promise as a support of in vitro ovarian follicular culture. HAM was decellularized with trypsin and EDTA. DNA and histology assays were performed to determine the elimination rate of genomic components. Cyto-biocompatibility of decellular AM (DAM) was verified by the cell viability (MTT) test. The small parts of intact amniotic membrane (IAM) and DAM were coated on the bottom of 96-well and each well was filled with 150 µL of base medium. Mouse primary-secondary (PS) follicles were separated to three groups: 1-culture in base medium (Control), 2-culture on IAM and 3-culture on DAM. Follicular size, morphology, viability, estradiol production and genes expression were evaluated and IAM group showed better growth and development in follicle culture. The viability rate and estradiol production in both experimental groups were statistically higher than the Control. Gdf9, Bmp15 and Cx37 were found to have higher expression levels in IAM group. Also, maximum apoptotic and survival indexes were determined in Control and IAM groups, respectively. Finally, IAM provides a better protective environment for mouse PS follicular culture that can reduce apoptosis level.

A low-cost mesocosm for the study of behaviour and reproductive potential in Afrotropical mosquito (Diptera: Culicidae) vectors of malaria.

A large-scale mesocosm was constructed and tested for its effectiveness for use in experiments on behaviour, reproduction and adult survivorship in the Afrotropical malaria vector Anopheles gambiae s.s. Giles (Diptera: Culicidae) in temperate climates. The large space (82.69 m(3) ) allowed for semi-natural experiments that increased demand on a mosquito's energetic reserves in an environment of widely distributed resources. A one-piece prefabricated enclosure, made with white netting and vinyl, prevented the ingress of predators and the egress of mosquitoes. Daylight and white materials prompted the mosquitoes to seclude themselves in restricted daytime resting sites and allowed the easy collection of dead bodies so that daily mortality could be assessed accurately using a method that accounts for the loss of a proportion of bodies. Here, daily, age-dependent mortality rates of males and females were estimated using Bayesian Markov chain Monte Carlo simulation. In overnight experiments, mosquitoes successfully located plants and took sugar meals. A 3-week survival trial with a single cohort demonstrated successful mating, blood feeding, oviposition and long life. The relatively low cost of the mesocosm and the performance of the mosquitoes in it make it a viable option for any behavioural or ecological study of tropical mosquitoes in which space and seasonal cold are constraining factors.

Improvement in in vitro fertilization rate, decrease in reactive oxygen species and spermatozoa death incidence in rams by dietary fish oil.

Our aim was to evaluate the effects of fish oil feeding on sperm classical parameters, level of reactive oxygen spices (ROS), spermatozoa death incidence and in vitro fertilization (IVF) rate in rams. We randomly assigned nine rams, into two experimental groups (isoenergetic and isonitrogenous rations with constant level of vitamin E supplement): control (CTR; n = 5) and fish oil (FO; n = 4, 35 g/day/ram). Diets were fed for 70 days during the physiological breeding season. After a 21-day dietary adaptation period, semen was collected weekly from each ram by an artificial vagina. Sperm classical parameters were determined by the computer-assisted sperm analyzer system (CASA), and it was prepared for IVF process by swim-up technique. These evaluations were performed during the first and last weeks of sampling. Intracellular ROS level and spermatozoa death incidence were detected by flow cytometry on a weekly basis after adaptation. Data were analysed with SPSS 15. The volume, concentration (3.6 and 2.7 × 10(9) /ml) and sperm progressive motility (60 and 48%) were significantly improved in the FO group compared with the CTR (p < 0.05). A comparison of two-cell stage embryos following IVF in the two groups showed a significantly higher fertilization rate in the FO group (56%) compared with the CTR (49%). Superoxide anion (O2 (-) ) rate was significantly lower (p < 0.05) at the third week of sampling in the FO. Although the H2 O2 rate was numerically lower in the FO group compared with the CTR, this difference was not significant. In addition, apoptosis showed a significant difference in the third week of sampling (15 and 30% for FO and CTR, respectively; p < 0.05). Overall, adding fish oil to the ram diet not only improved sperm quality and IVF results, it also could reduce oxygen-free radicals and the incidence of spermatozoa death.

Effect of cysteine and glutamine added to extender on post-thaw sperm functional parameters of buffalo bull.

Amino acids seem to be crucial components for semen freezing extender due to antioxidant properties. Therefore, this study aimed to assess motility parameters, membrane integrity, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and DNA damage to detect the optimum concentrations of cysteine and glutamine for buffalo semen cryopreservation. Twenty ejaculates of four buffalo bulls were diluted in tris-egg yolk extender and divided into seven equal groups consisting of cysteine (5, 7.5 and 10 mmol), glutamine (10, 15 and 20 mmol) and no additive. Supplementation of 5 and 7.5 mmol cysteine and 15 mmol glutamine in cryopreservation extender significantly increased post-thaw motility and plasma membrane integrity of spermatozoa with significant reduction in intracellular ROS when compared with control groups (P < 0.05). Cysteine at 7.5 mmol concentration elevated progressive motility and MMP, compared with control (P < 0.05). No significant differences were observed for motion patterns and DNA damage of frozen-thawed buffalo spermatozoa in extender containing amino acids. The findings of this study showed that supplementation of 7.5 mmol cysteine and 15 mmol glutamine in semen cryopreservation extender has more potential to decrease intracellular ROS, and subsequently elevate motility and membrane integrity of buffalo frozen-thawed spermatozoa.

Expression of maturation genes and their receptors during in vitro maturation of sheep COCs in the presence and absence of somatic cells of cumulus origin.

The purpose of this study was to investigate the effects of somatic cells of cumulus origin (sCC) on gene expression and maturation of cumulus oocyte complexes (COCs) in vitro. Good quality (i.e., healthy-looking) isolated sheep COCs were randomly divided into two treatment groups: control (COC with no sCC) and coculture (COC with sCC). Nuclear maturation statuses of oocytes were assessed after 27 hours of in vitro culture. Moreover, the expression levels of growth differentiation factor 9 (GDF9), bone morphogenetic protein (BMP)15, BMP6, bone morphogenetic protein receptor II (BMPRII), activin like kinase 5 (ALK5) (transforming growth factor ß receptor 1: TGFßR1), ALK6 (BMPR1b), activin A receptor, type IIB (ActRIIB), and ALK3 (BMPR1a), as well as hyaluronan synthase 2 (HAS2) and prostaglandin endoperoxide synthase 2 (Ptgs2) in the COCs were assessed in both treatment groups after 3 h and 27 h of culture. The results showed that the proportion of metaphase II (MII) stage oocytes was significantly higher in the coculture group compared with the controls (77.21%±1.17 vs. 67.49%±1.80; P<0.05). The relative expressions of BMPRII, ALK6, and ActRIIB in control group and GDF9 and ActRIIB in coculture group showed significant differences during culture as assessed by real time polymerase chain reaction (P<0.05). The mean expression levels of BMPRII, ALK5, ALK6, and ActRIIB mRNA were decreased in the coculture group compared with those in the control group after 27 h of culture (P<0.05). In conclusion, we propose that in vitro maturation of sheep COCs alone disrupted the normal gene expression levels of both TGFß ligands and receptors, and also reduced the maturation rate. Coculture with sCC enhanced the maturation rate of oocytes concomitantly with reduced gene expression levels of a number of TGFß ligands and receptors.

Most human metapneumovirus and human respiratory syncytial virus in infant nasal secretions is cell free.

BACKGROUND: Nasopharyngeal secretions aspirated from infants with bronchiolitis (NPA) are a valuable resource for the study of virus dynamics and local inflammatory responses, however samples are small and difficult to manipulate. OBJECTIVES: To improve yield of NPA from infants. To establish if removal of the cellular component of NPA affects quantification of human metapneumovirus (hMPV) or human respiratory syncytial virus (hRSV) genome. STUDY DESIGN: Weight of NPA collected into traps from 30 infants was compared with that collected in trap plus catheter and washed through with saline from another 30 infants. hMPV (n=33) and hRSV (n=30) genome was measured by reverse-transcribed real-time polymerase chain reaction (RT-RT-PCR) in paired whole and cell-free NPA collected by the improved method. RESULTS: The improved method of NPA collection gave near two-fold greater weight (p = 0.002) of NPA (mean = 0.52 g (S.D. = 0.30 g)) than the traditional method (0.32 g (S.D. 0.19)). There was strong agreement and no significant difference between viral load measured in whole and cell-free fractions of NPA for both viruses (samples (n), correlation coefficient (cc) and significance (p)); hMPV (n=33, cc=0.938, p<0.001) and hRSV (n=30, cc=0.977 and p<0.001). CONCLUSIONS: The majority of hRSV and hMPV in nasal secretions is not associated with cells. Removal of the cellular component of NPA does not interfere with quantification of hRSV and hMPV.

Epidemiological study in a new focus of cutaneous leishmaniasis in the Islamic Republic of Iran.

An outbreak of cutaneous leishmaniasis (CL) in Sabzevar county prompted this study of the epidemiology and the ecology of vectors and reservoirs. Examination of 541 schoolchildren showed rates of 9.4% for scars and 5.9% for ulcers. Among 807 inhabitants of 4 villages, 10.4% had scars and 3.0% had active lesions. The most highly infected age group was 0-4 years with a rate of 5.9%. A total of 12 849 sandflies representing 7 species were collected in the study area. Leptomonad infection was found in Phlebotomus papatasi, P. caucasicus and Sergentomyia sintoni. Parasites from man, P. papatasi and Rhombomys opimus, were isolated and characterized as Leishmania major. Based on this survey, this is an epidemic of zoonotic CL, with R. opimus the main reservoir host, and P. papatasi the main vector.

Epidemiological study in a new focus of cutaneous leishmaniasis in the Islamic Republic of Iran

An outbreak of cutaneous leishmaniasis [CL] in Sabzevar county prompted this study of the epidemiology and the ecology of vectors and reservoirs. Examination of 541 schoolchildren showed rates of 9.4% for scars and 5.9% for ulcers. Among 807 inhabitants of 4 villages, 10.4% had scars and 3.0% had active lesions. The most highly infected age group was 0-4 years with a rate of 5.9%. A total of 12 849 sandflies representing 7 species were collected in the study area. Leptomonad infection was found in Phlebotomus papatasi, P. caucasicus and Sergentomyia sintoni. Parasites from man, P. papatasi and Rhombomys opimus, were isolated and characterized as Leishmania major. Based on this survey, this is an epidemic of zoonotic CL, with R. opimus the main reservoir host, and P. papatasi the main vector

Murine gammaherpesvirus-68 infection causes multi-organ fibrosis and alters leukocyte trafficking in interferon-gamma receptor knockout mice.

Murine gammaherpesvirus-68 (MHV-68) infection in interferon-gamma receptor knockout mice (IFN-gammaR(-)/(-)) results in splenic fibrosis and excessive loss of splenocytes. In our present study we found that MHV-68 infection in IFN-gammaR(-)/(-) mice also resulted in fibrosis and atrophy of the mediastinal lymph nodes, interstitial pulmonary fibrosis and fibrotic changes in the liver. Atrophy and cellular depletion of the spleen in IFN-gammaR(-)/(-) was not the result of increased cell death. The loss of splenocytes in IFN-gammaR(-)/(-) mice, which was most evident on day 23 after infection, correlated with an increase in the number of leukocytes in peripheral blood. At the peak of leukocytosis, on day 23 after infection, peripheral blood cells from infected IFN-gammaR(-)/(-) mice were unable to traffic through the fibrosed spleens of IFN-gammaR(-)/(-) mice but were able to enter the spleens of wild-type mice. This indicates that leukocytosis was in part the result of emigration of cells from the spleen and their subsequent exclusion of re-entry at the height of fibrosis. Significant cytokine and chemokine changes were observed in spleens of IFN-gammaR(-)/(-) mice. IFN-gamma, tumor necrosis factor-alpha (TNF-alpha ), TNF-beta, interleukin-1beta (IL-1beta), transforming growth factor-beta1 (TGF-beta1), lymphotactin, and MIP-1beta were elevated on day 14 after infection whereas chemokines IP-10 and MIG were significantly reduced. These changes suggest a role for dysregulated cytokines and chemokines in severe organ-specific fibrosis with implications for immune-mediated fibrotic disorders.

Phenotypic characterisation and infection of ovine microglial cells with Maedi-Visna virus.

Maedi-Visna Virus (MVV) infection of the central nervous system (CNS) results in pathological changes, the mechanisms of which are poorly understood. MVV preferentially infects cell of the monocyte/macrophage lineage in vivo. The neuroparenchymal microglial cells are the resident tissue macrophages in the CNS and therefore likely targets for MVV infection. However, no information is currently available on the susceptibility of these cells to MVV infection or their contribution to neuropathological changes as a result of MVV infection. Highly enriched primary ovine microglial cell cultures were set up from brain tissues of lambs. These cells were amoeboid or bipolar with spikes, a morphology consistent with microglial cells of other species, and stained positive for CD1, CD11a, CD11c, CD14, MHC-class I, MHC-class II, and beta-N-acetyl galactose, but not with markers of astrocytes or oligodendrocytes. These sheep microglial cells were permissive for MVV infection. Productive MVV infection resulted in selective transcriptional up-regulation of the pro-inflammatory cytokines TNFalpha and IL-6. In contrast, there was no change in levels of transcripts for TGFbeta1, IL-1beta, GM-CSF, IL-10, or IL-12. These data provide the first evidence that ovine microglial cells can support productive infection with MVV, and that this leads to a selective up-regulation of proinflammatory cytokines. These may contribute to visna neuropathology.

Murine gammaherpesvirus M11 gene product inhibits apoptosis and is expressed during virus persistence.

The murine gammaherpesvirus (MHV-68) M11 gene encodes a protein with BH1 domain homology to Bcl-2. We found that the M11 gene product (MHVBcl-2) protected murine epithelial cells from TNF-alpha induced apoptosis. M11 was transcribed during early lytic infection in vitro. During early infection of mice, M11 message was detected in spleen and lung along with lytic cycle messages. During persistence, lytic cycle gene expression was undetectable but M11 RNA was still present. This suggests that MHVBcl-2 promotes virus survival by protecting not only productively infected but also persistently infected cells from apoptotic death.

Novel cell lines for the analysis of preprotachykinin A gene expression identify a repressor domain 3' of the major transcriptional start site.

Until now, no clonal cells have been identified that support the expression of a marker gene expressed from the rat preprotachykinin A (rPPT) promoter. We have analysed recently available cell lines that are candidates for supporting reporter gene expression directed by the rPPT promoter. These are the neuronal-derived cell line NF2C and the pancreatic cell lines RINm5F and a derivative RIN-1027-B2. The NF2C line was derived from the brain homogenate of a transgenic animal in which a temperature-sensitive simian virus 40 large T antigen was expressed from a neurofilament promoter. All three lines are able to support expression of a reporter gene directed by a fragment of the 5' rPPT promoter. Analysis of reporter gene expression supported by various fragments of the rPPT promoter demonstrated that although -865 to +92 bp supported expression, the addition of fragments between +92 and +447 bp led to repression of expression. Subsequent analysis of reporter gene constructs microinjected into primary cultures of dorsal root ganglion neurons (DRG) confirmed the existence of this repressor domain. This repression could be relieved totally in both RIN cell lines and partly in NF2C cells by mutating residues between +373 and +396 bp. This indicates that these cell lines support PPT promoter activity similar to that observed in DRG and determines a novel repressor domain within the promoter.

Paracrine induction of stem cell renewal by LIF-deficient cells: a new ES cell regulatory pathway.

The propagation of pluripotential mouse embryonic stem (ES) cells is sustained by leukemia inhibitory factor (LIF) or related cytokines that act through a common receptor complex comprising the LIF receptor subunit (LIF-R) and the signal transducer gp130. However, the findings that embryos lacking LIF-R or gp130 can develop beyond gastrulation argue for the existence of an alternative pathway(s) governing the maintenance of pluripotency in vivo. In order to define those factors that contribute to self-renewal in ES cell cultures, we have generated ES cells in which both copies of the lif gene are deleted. These cells showed a significantly reduced capacity for regeneration of stem cell colonies when induced to differentiate, confirming that LIF is the major endogenous regulatory cytokine in ES cell cultures. However, self-renewal was not abolished and undifferentiated ES cell colonies were still obtained in the complete absence of LIF. A differentiated, LIF-deficient, parietal endoderm-like cell line was derived and shown to support ES cell propagation via production of a soluble, macromolecular, trypsin-sensitive activity. This activity, which we name ES cell renewal factor (ESRF), is distinct from members of the IL-6/LIF family because (i) it is effective on ES cells lacking LIF-R; (ii) it is not blocked by anti-gp130 neutralizing antibodies; and (iii) it acts without activation of STAT3. ES cells propagated clonally using ESRF alone can contribute fully to chimaeras and engender germline transmission. These findings establish that ES cell pluripotency can be sustained via a LIF-R/gp130-independent, STAT-3 independent, signaling pathway. Operation of this pathway in vivo could play an important role in the regulation of pluripotency in the epiblast and account for the viability of lifr -/- and gp130 -/- embryos.

Cloning, sequencing and expression of the ovine interleukin 6 gene.

Gene amplification by reverse transcriptase PCR with heterologous primers has been used to obtain a cDNA clone encoding the structural sequences of ovine interleukin 6 from alveolar macrophages. This cDNA encodes a protein of M(r) = 23,429, which is 53% homologous in amino acid sequence to human IL = 6. The clone hybridizes to an RNA of size 1260 nt in alveolar macrophages, expression of which is potentiated by LPS. The ovine IL-6 structural gene has been cloned into the yeast expression vector pOGS40, and used to produce a recombinant protein. This protein is capable of causing increased immunoglobulin production in pokeweed mitogen stimulated ovine peripheral blood mononuclear cells at concentrations of 10-100 ng/ml, but it only causes very limited replication of B9 cells, a murine IL-6 dependent cell line. This is in contrast to recombinant human IL-6, which is capable of stimulating B9 cell proliferation, but not immunoglobulin production by ovine PBMC.