We are unceasingly exposed to potentially harmful microorganisms. The battle against threatening infectious agents includes activation of both the innate and of the adaptive immune systems. Illness responses are elicited and include inflammation, fever, decreased appetite, lethargy and increased sensitivity to painful stimuli in order to defeat invaders. While many of these signs of disease are controlled by the central nervous system, it has remained an enigma how signals from the peripheral immune system reach the brain through its blood-brain barrier, which precludes macromolecules, including cytokines, from diffusing into the brain parenchyma. Previous findings indicate the existence of a pathway across the blood-brain barrier, which includes binding of the cytokine interleukin-1 (IL-1) to its receptor in the brain vessels, thereby inducing the production of the prostaglandin E2 (PGE2) synthesizing enzymes cyclooxygenase-2 (Cox-2) and microsomal prostaglandin E synthase-1 (mPGES-1), which ultimately synthesize PGE2. PGE2 subsequently binds to any of the four prostaglandin E2 (EP) -receptors. Previous results from our laboratory have suggested that this pathway plays a critical role in the febrile response to infectious stimuli. The present thesis aims at further investigating the molecular events underlying immune-to-brain signalling, with special emphasis on fever, hypothalamic-pituitary-adrenal (HPA) -axis activation and anorexia and their connection to signalling molecules of the cytokine and prostaglandin families, respectively. In paper I, the molecular processes linking the proinflammatory cytokine interleukin-6 (IL-6) and PGE2 in the febrile response were investigated. Both IL-6 and PGE2 have been shown to be critical players in the febrile response, although the molecular connections are not known, i.e. if IL-6 exerts its effects up- or downstream of PGE2. Mice deficient in IL-6 were unable to respond to bacterial lipopolysaccharide (LPS) with a febrile response, but displayed similar induction of Cox-2 and mPGES-1, and similar concentrations of PGE2 in the cerebrospinal fluid as wild-type mice. Paradoxically, the IL-6 deficient mice responded with a dose-dependent elevation of body temperature in response to intracerebroventricularly injected PGE2. Furthermore, IL-6 per se was not pyrogenic when injected peripherally in mice, and did not cause increased levels of PGE2 in cerebrospinal fluid. IL-6 deficient mice were not refractory to the action of PGE2 because of excess production of some hypothermia-producing factor, since administration of a Cox-2 inhibitor in LPS-challenged IL-6 deficient mice did not unmask any hypothermic response, and neutralization of tumor necrosis factor α (TNFα), associated with hypothermia, did not produce fever in LPS-challenged IL-6 deficient mice. These data indicate that IL-6 rather than exerting its effects up- or down-stream of PGE2 affects some process in parallel to PGE2, perhaps by influencing the diffusion and binding of PGE2 onto its target neurons. In papers II and III, we injected the proinflammatory cytokine IL-1β in free-fed wild-type mice, in mice with a deletion of the gene encoding mPGES-1, or in mice deficient in the EP1, EP2 and EP3. Food intake was continuously measured during their active period, revealing that mPGES-1 deficient mice were almost completely resistant to anorexia induced by IL-1β. However, all of the investigated EP receptor deficient mice exhibited a normal profound anorexic response to IL-1β challenge, suggesting that the EP4 is the critical receptor that mediates IL-1β-induced anorexia. We also investigated the role of mPGES-1 in anorexia induced by lipopolysaccharide (LPS) in mPGES-1 deficient mice. The profound anorexic response after LPS-challenge was similar in mPGES-1 deficient and wild-type mice. To further investigate the anorectic behaviour after LPS injection, we pre-starved the animals for 22 hours before injecting them with LPS. In this paradigm, the anorexia was less profound in mPGES-1 knock-out mice. Our results suggest that while the inflammatory anorexia elicited by peripheral IL-1β seems largely to be dependent on mPGES-1-mediated PGE2 synthesis, similar to the febrile response, the LPS-induced anorexia is independent of this mechanism in free-fed mice but not in pre-starved animals. In papers IV and V, the role of prostanoids for the immune-induced HPA-axis response was investigated in mice after genetic deletion or pharmacological inhibition of prostanoid-synthesizing enzymes, including Cox-1, Cox-2, and mPGES-1. The immediate LPS-induced release of ACTH (adrenocorticotropic hormone and corticosteroids was critically dependent on Cox-1 derived prostanoids and occurred independently of Cox-2 and mPGES-1 derived PGE2. In contrast, the delayed HPA-axis response was critically dependent on immune-induced PGE2, synthesized by Cox-2 and mPGES-1, and occurred independently of Cox-1 derived enzymes. In addition, in the mPGES-1 deficient mice, the synthesis of CRH hnRNA and mRNA was decreased in the paraventricular nucleus of the hypothalamus after LPS-challenge, indicating that the delayed hormone secretion was mediated by PGE2-induced gene-transcription of CRH in the hypothalamus. The expression of the c-fos gene and Fos protein, an index of synaptic activation, was maintained in the paraventricular nucleus and its brainstem afferents both after unselective and Cox-2 selective inhibition as well as in Cox-1, Cox-2, and mPGES-1 knock-out mice. This suggests that the immune-induced neuronal activation of autonomic relay nuclei occurs independently of prostanoid synthesis and that it is insufficient for eliciting stress hormone release.

Swine barn air is a heterogeneous mixture of dust, bacteria and irritant chemicals including ammonia and hydrogen sulphide. Gram-negative bacteria are commonly found in swine barn air and significantly contribute to pulmonary disease in unprotected swine barn workers, through the endotoxin moiety, lipopolysaccharide (LPS). Toll-like Receptor-4 is the ligand for LPS. It is found on many cell types including monocytes, macrophages, neutrophils, endothelial cells, and to a lesser extent, epithelial cells. The severity and outcome of acute lung injury following barn air exposures depends upon the balance between epithelial and vascular endothelial repair mechanisms, including angiogenesis. Vascular Endothelial Growth Factor (VEGF) is an endothelial mitogen produced by mesenchymal and alveolar Type II epithelial cells and by activated bronchial airway epithelial cells. Research investigating the role of cytokines in angiogenesis has shown that close proximity of immune cells and endothelial cells modulates the production of various compounds that regulate vascular function. Given that LPS is the ligand for TLR4 there appeared to be a role for TLR4 in angiogenesis, particularly following endotoxin exposure. To determine whether this was occurring, we examined whether exposure to swine barn air alters vascular density in the lungs and the role of TLR4 using a murine model. Toll-like Receptor-4 wild-type (C3HeB/FeJ) and TLR4 mutant (C3H/HeJ) mice were obtained and exposed to swine barn air for 1-, 5-, or 20-days for 8 hours/day. Wild-type animals showed a 127% increase in vascular density after 20-days barn air exposure. Vascular Endothelial Growth Factor-A protein levels were decreased by 0.62-fold after one-day swine barn air exposure in wild-type animals, indicating that VEGF-A is being used as a pro-angiogenic mitogen. Transcription of VEGF-A mRNA was increased in wild-type animals after all swine barn air exposure periods. The receptor VEGFR-1 showed increased mRNA transcription over all time points. These effects were only observed in TLR4 wild-type animals, indicating that these effects are mediated by TLR4. Further, VEGF-A and VEGFR-1 appear to be involved in the manifestation of TLR4-induced angiogenesis in the lung.

Swine barn air is a heterogeneous mixture of dust, bacteria and irritant chemicals including ammonia and hydrogen sulphide. Gram-negative bacteria are commonly found in swine barn air and significantly contribute to pulmonary disease in unprotected swine barn workers, through the endotoxin moiety, lipopolysaccharide (LPS). Toll-like Receptor-4 is the ligand for LPS. It is found on many cell types including monocytes, macrophages, neutrophils, endothelial cells, and to a lesser extent, epithelial cells. The severity and outcome of acute lung injury following barn air exposures depends upon the balance between epithelial and vascular endothelial repair mechanisms, including angiogenesis. Vascular Endothelial Growth Factor (VEGF) is an endothelial mitogen produced by mesenchymal and alveolar Type II epithelial cells and by activated bronchial airway epithelial cells. Research investigating the role of cytokines in angiogenesis has shown that close proximity of immune cells and endothelial cells modulates the production of various compounds that regulate vascular function. Given that LPS is the ligand for TLR4 there appeared to be a role for TLR4 in angiogenesis, particularly following endotoxin exposure. To determine whether this was occurring, we examined whether exposure to swine barn air alters vascular density in the lungs and the role of TLR4 using a murine model. Toll-like Receptor-4 wild-type (C3HeB/FeJ) and TLR4 mutant (C3H/HeJ) mice were obtained and exposed to swine barn air for 1-, 5-, or 20-days for 8 hours/day. Wild-type animals showed a 127% increase in vascular density after 20-days barn air exposure. Vascular Endothelial Growth Factor-A protein levels were decreased by 0.62-fold after one-day swine barn air exposure in wild-type animals, indicating that VEGF-A is being used as a pro-angiogenic mitogen. Transcription of VEGF-A mRNA was increased in wild-type animals after all swine barn air exposure periods. The receptor VEGFR-1 showed increased mRNA transcription over all time points. These effects were only observed in TLR4 wild-type animals, indicating that these effects are mediated by TLR4. Further, VEGF-A and VEGFR-1 appear to be involved in the manifestation of TLR4-induced angiogenesis in the lung.

This study developed a summative scale that could be administered in a short time period to determine the Quality of Use (QOU) of an intervention used by teachers. The scale can be completed in less than an hour using easily attainable information. The QOU scale was applied to an elementary reading program to determine if the program results were dependent upon the quality of the fidelity of teacher use. The study focused on use of the Linguistic Pattern Series (LPS) portion of the Integrated Skills Method (ISM) Reading Program by 20 special education teachers in 13 elementary schools in San Antonio, Texas. Progress is measured by the use of the Decoding Skills Test (DST) (ISM Teaching Systems, Inc., 2004-b). To determine each teacher's QOU, a summative scale was developed composed of five items (Initial Placement, Frequency of Direct Instruction, Materials – LPS, Materials – Literature/Test Prep, and Scheduling) that possessed low-moderate cohesiveness of Alpha=.71. Results of the study showed a correlation between QOU summary scores and residualized DST Raw Score Grade Equivalent (RSGE) gains. The QOU could predict .771^2 = 50 percent of score variance. This is a strong prediction for a non-student external measure in education.

Lightning is a natural phenomenon involving transient high current discharge in the atmosphere. Cloud-to-ground lightning, wherein the discharge occurs between the cloud and the ground is quite hazardous to systems on the ground. Apart from threat to life, the devastating effects of lightning can be mainly of thermal, mechanical and electromagnetic origin. Many a times, thermal and electromagnetic effects are of main concern. A direct hit, wherein the system under consideration becomes a part of the lightning path, could be quite catastrophic to many vulnerable systems like oil rigs, chemical factories, missile/satellite launch pads. From the safety and operational point of view, lightning is of serious concern for electrical systems including transmission lines and substations, nuclear power stations, telecommunication station and data banks. Lightning cannot be avoided, however, by employing a suitable Lightning Protection System (LPS), adequate protection against a direct hit can be provided to ground based systems. A typical lightning protection system involves: 1) Air termination network, which is responsible for stroke interception, 2) Down conductor system, which provides to the stroke current a minimal impedance path to the ground and 3) Earth termination network, for safe dissipation of current into the ground. Similarly, for the indirect effects, which are basically of electromagnetic origin, suitable protection can be designed. The key factors in a protective action involve interception of the dangerous strokes, minimization of the consequential potential rise on down conductors, as well as, at earth termination and keeping the field in the protective volume within an acceptable level. The last aspect can be generally categorized into secondary level protection. For critical systems, the lightning protection system is generally isolated from it. In such designs, potential rise on LPS governs the physical isolation required between the protected and protection system. For a given level of bypass strokes, cost of the LPS increases with the amount of physical separation employed. All most all of the earlier works have concentrated on lightning surge response of power transmission line towers. Apart from their relatively moderate heights, the intention was to arrive at a model, which can be incorporated in circuit simulation software like EMTP. Consequently, they envisage or approximate the mode of propagation to be TEM. In reality, for down conductors of height greater than say 30 m, only TM mode prevails during the initial critical time period. Hence the earlier models cannot be extended to general lightning protection schemes and for down conductor of larger lengths. Only limited literature seems to be available on the characteristics of general down conductor configurations. The problem in hand is very important and some serious research efforts are very much essential. In view of the above, the present work aims to evaluate the rise in potential as well as current injected into the soil at the base for: (i) practical range of down conductor configurations involving single down conductor (with height exceeding 30 m) and (ii) pertinent values of stroke current parameters. The protection schemes considered are isolated vertical down conductor, isolated tower (both square and triangular cross-section) and, tower with insulated lightning mast carrying ground wires. The parameters under consideration are: (i) height and cross section for the down conductor, (ii) clearance between the down conductor and the protected system, (iii) channel geometry, wherein only inclination is to be considered, (iv) velocity of current along the channel and (v) wave shape and rise time for the stroke current. For the evaluation of lightning surge response of transmission line towers, many theoretical and experimental approaches are found in the literature. However, works considering the TM mode of current propagation is relatively limited. In that both experimental and theoretical approaches have been adopted. Theoretical approach invariably adopted numerical field computation in frequency domain using Numerical Electromagnetic code (NEC-2). Fourier Transform techniques are employed to extract the time domain quantities. This approach is very economical, free from experimental errors and least time consuming. Hence it is selected for the present work. However, there are certain limitations in this approach. In NEC simulation, there is a restriction on the size and the arrangement of individual elements. Therefore, although fairly complex tower structures can be simulated, some simplification in the geometry is unavoidable. Such an approximation has been reported to cause insignificant error. NEC is not accurate for calculations in low frequency regime. But in the present work, the initial time regime is of concern wherein the high frequency components dominate. Therefore the above said limitation is not of any serious concern. In order to validate the approach, potential rise is computed for 120 m tall cylindrical down conductor and tower. Results are compared favorably with earlier works, which are based on potential lead wire method. A careful re-look into the ’potential rise’ on the down conductors reveal several things. The electric field in the region between the protection system and protected system is the root cause for the breakdown/flashover. For a given geometry, the integral of the electric field along the shortest path between the two systems must be representing the overall stress on the air gap. Further, for the later time periods, this integral coincides with the well-known quasi-static potential. All the available data and models for breakdown of long air gaps are basically in terms of this quasi-static potential. In view of this, the above path integral is defined as ’equivalent potential rise’ (which will be hereafter termed as ’potential rise’), and taken as the index for surge response. Further, observation of the computed spatio-temporal radial electric field around the down conductor reveals some additional features, which are not common in the quasi-static regime. Electric field reverses its polarity in space, which is due to the opposite current flowing in the lightning channel. Therefore, ’potential rise’, which is taken as the representative for the dielectric stress on the air, should not be evaluated for larger distances. Considering this and noting that the protected system generally lies well within a distance of 50% of the H, height of the down conductor, potential rise is evaluated by integrating electric field within this distance (12.5%H, 25%H, 50%H). Three heights (100%H, 75%H, 50%H) are considered for the evaluation of the potential. The influences of various down conductor and lightning channel parameters are analyzed. Finally vertical channel with full velocity for current propagation is arrived for the investigations. Also, the influence of neighboring conducting objects is briefly studied. It is argued that it needs to be ignored for the general study. Analysis is carried out for a range of down conductor configurations of heights ranging from 45 m to 120 m. Cylindrical down conductor is selected for the detailed study on the overall characteristics and its dependency on pertinent parameters. The characteristics of potential rise are found to be significantly different from that given by the commonly employed uniform transmission line model. In the regime of very fast front currents, down conductor of comparable heights have comparable potential rise. For the larger time to crest, behavior tends more to wards that for quasi-static regime. The dependency of the potential rise on radius of the down conductor seems to be logarithmic in nature. Surge response of isolated towers of both square and triangular cross sections is studied for heights ranging from 45 m to 120 m. The overall characteristics are found to be similar to cylindrical down conductor. Dispersive propagation is found to exist on towers. As a result, the base currents are slightly lower and potential rise exhibits less oscillations. Data curves on potential rise at three different heights and for three different spatial extents are generated for the range of down conductor heights with rise time of the stroke current as the variable. Several interesting observations have been made. Next the investigation is taken up for the insulated mast scheme. The parameters of the study are taken as the number of ground wires, grounding location of ground wires and length of the insulation cylinder. Potential across the insulation, tower base currents, and ground wire end currents are deduced. The basic characteristics of the potential rise are shown to be quite similar to that for the transmission line. For fast front currents the temporal variation is bipolar with a smooth decay. In other words, oscillations are sustained for considerably longer duration. Voltage stress across the insulation surface for one ground wire design is found to be higher by 1.4 - 2.4 times than that for isolated tower. The highest amplification of the ground end current, which occurs for fast front currents, is about 1.8 times. Potential difference across the insulation for two-ground wire design is higher by a factor of 1.3 - 1.85 than that for isolated tower. For the design with four ground wires, potential across the insulation is comparable with that for the tower. However, the mechanical strength of the insulating support should also be considered in the selection of number of ground wires. There exists, especially for fast front strokes, significant induction to the supporting tower. The height of the insulation seems to possess no appreciable influence on the potential rise and base currents. Several issues need to be considered before selecting this design. The contribution made by the present work can be summarized as follows. It basically deals with lightning surge response of isolated down conductors of height in the range 45 - 120 m. The configurations considered are, cylindrical down conductor, tower with both square and triangular cross section and insulated mast scheme. It makes a careful study on the ’potential rise’ on down conductors and a suitable definition for the same is proposed. Basic characteristics of potential rise and ground end currents are studied for the above-mentioned designs. Their salient features are enumerated. For the towers, design data curves are provided for relevant range of stroke current rise time. The issues that need to be considered in the insulated mast scheme are discussed along with the data on potential rise and base currents. The findings of this work are believed to be very useful for the design of lightning protection scheme involving isolated down conductor. Further the results are useful in analyzing the consequential lightning generated threat of being close to tall towers.

Satellites have become absolute necessity in the growing modern space technology. At present, launch pads are the only means for launching of satellites or any other space vehicles. Due to the large magnitude of current and the associated rate of rise, a lightning strike to launch pads can be quite disastrous. Satellite launch complex forms typically the tallest object in that region. This makes them the more vulnerable to cloud-to-ground lightning. In addition, most of the launch pads are situated near the coastal area, where the isokeraunic levels are quite high. In view of these, almost all the satellite launch pads are provided with suitable Lightning Protection Systems (LPS). The LPS is basically intended for protecting against a direct lightning hit. The present work is related with the LPS to Indian satellite launch pads, Pad-I and Pad-II. The protection system for Pad-I consists of three 120 m tall towers placed approximately at the vertices of an equilateral triangle of 180 m. The same for Pad-II consist of 120 m tall towers placed at vertices of rectangle of size 90 m x 105 m. Towers are interconnected by 6 shield wires at the top. A mast of 10m length forms the top of the tower. Significant work on the analysis of interception efficacy of these protection systems has been reported in the literature. The lightning surge response of these systems have also been analysed and reported. The interception efficacy of these LPS in field can be ascertained by pertinent measurements. Measuring the lightning current on LPS seems to be one of the most suitable choices for this purpose. It would also greatly facilitate collection of local lightning current statistics, data on which is almost absent. Several considerations suggest that the tower bases form ideal place for such measurement. However, such lightning current records would involve mainly the current resulting from stroke interception, as well as, induced current due to strokes nearby. Literature on categorisation of measured currents to the type of stroke and correlation of measured currents to the incident stroke currents is rather limited. This is especially true for interconnected protection system of the type dealt in the present work. Considering these the present work is taken up and its scope is defined as: (i) Evolve a suitable model for study of current distribution in LPS due to Lightning and using the same deduce the current due to stroke interception and that due to stroke nearby. (ii) For the purpose of categorization identify the salient characteristics of current due to the intercepted strokes and that due to bypass/nearby strokes (iii) For the intercepted strokes, develop a processor for estimating the injected stroke current from the measured tower base currents. Lightning event, apart from other associated physical phenomena, is strongly governed by electromagnetic fields. Any method employed for the analysis, either theoretical or experimental, should satisfy the governing electromagnetic equations. As experimentation on actual system, as well as, their laboratory simulation is nearly impossible, theoretical modelling approach is selected. Modelling involves modelling of the channel along with its excitation, modelling of the LPS and modelling of the ground. Channel, following the literature, is represented as a loaded conductor with a lumped current source at the junction point. Such models have quite successfully predicted the electromagnetic fields and current in other places on the down conductor. For the LPS, some simplifications on the geometry are very essential. Tower lattice elements of dimensions much smaller than the wavelength of highest dominant frequency component of lightning current spectrum are neglected. Suitable modification is made for the tower top involving a plate and interconnection of several short members. For the close range within 200 – 400 m, even for the induced currents, the influence of ground in the literature has been reported to be small. Also, there is an extensive grounding network in these systems. In view of the same, a perfectly conducting ground along with suitable ground termination impedance is considered. Only the numerical solution of the problem is feasible and for the same, following the literature, NEC-2 is employed. All the guidelines of NEC are respected in the discretisation. Geometric mean radius is employed for modelling the complex tower elements. Fourier Transform Techniques are employed for time domain conversion of the computed frequency domain quantities. Occasionally, numerical inversion error of magnitude less than 5% is encountered. For the validation of the numerical modelling for both direct stroke and that nearby, time domain experimentation on electromagnetically reduced scale models (35:1) is employed. As the channel electrical and geometrical parameters are stochastic in nature, it is necessary to ensure that the deduction made using the model is practically relevant. For this, some parametric studies are conducted. The influence of channel length and inclination, stroke current velocity etc. has been shown to be insignificant for the case of intercepted strokes. Simulations are carried out for the stroke intercepted (i.e. direct strikes) by the LPS. The characteristics of the tower base currents are investigated. The base currents indicate a dispersive propagation along the towers and further a frequency dependent current division at the tower-shield wire junctions. Base currents contain superimposed oscillations, which basically originate from various junctions of the system. The magnitude of the oscillations is obviously dependent on the rise time of the incident currents. The tower base currents settle within about 10 -15 µs, which is shorter than that for isolated tower. Further, the full-frequency model could be limited to this time period. The corresponding current transfer functions are deduced. For the stroke interception by shield wires, based on the earlier work, only stroke to midspan is found to be relevant and hence it is considered. The nature of tower base currents for a stroke to midspan of the shield wires seem to be similar. However there are some distinct features, which are helpful in identifying the stroke location on the LPS. From the time correlated tower base currents, a suitable methodology for identifying the stroke interception location on LPS is developed. Next, simulations for induced current due to a bypass stroke, as well as, stroke to ground outside the LPS, however, within 1 km radius are taken up. In fact, it is estimated that latter is nearly 5 – 13 times higher than the strokes collected by LPS, indicating it as the most probable event. The objective here is characterization, rather than correlation. In this study, the influence of charge induced on the LPS by the descending leader is neglected and the upward leader activity is approximately considered. To the best of author’s knowledge, studies on such induced currents in down conductors are very scarce. Considering this and noting that the number of parameters is quite large, first the basic study is taken up on simple cylindrical down conductors. Many important and interesting deductions are made. The nature of the induced current is highly dependent on the rate of rise as well as the velocity of propagation of the stroke current. The magnitude and to some extent, the wave shape of the induced current is found to depend on the average as well as maximum di/dt of the stroke current. For a given wave shape, the magnitude of the induced current increases with rate of rise of the wave front; however, saturating trend will onset after some point. The height of the down conductor mainly governs the frequency of the oscillatory component of the induced current. The dependency of the induced current on the radius of the down conductor seems to be logarithmic (which is in accordance with the antenna theory). Based on these results, the parameters for the corresponding study on LPS under consideration, is chosen. The results of the investigation on the induced currents in LPS show that they have quite distinct waveform. They are basically bipolar and oscillatory in nature, with relatively short duration. These unique features facilitate clear distinction of the induced currents from that due to stroke interception. Basic characteristics are reasonably insensitive to the separation distance of the protection system and the channel, current propagation velocity along the channel, channel inclination and shape of the current front. The salient features of the induced current due to a bypass stroke are also enumerated. • The noise, if any, in the measured current can be addressed only after acquiring sufficient data. Based on the above, the following procedure is suggested for the stroke classification and estimation. • By employing the distinct features of the resulting tower base currents, analyze the measured tower base currents and classify the strokes into the intercepted stroke or stroke to ground. • For the latter case, using the salient features of the bypass strokes, further classify the strokes to bypass strokes and stroke to ground outside the protected volume. • For the intercepted strokes, using the relative strengths and wave shapes, identify the interception point to either tower top or the midspan of the shield wires. • Then by using the corresponding transfer functions and Fourier Transform techniques, compute the injected stroke current. • Using the above, other tower base currents are computed and compared with the measured currents. This gives quantification for the accuracy of the method. In summary the present work has made some original contribution to the classification and estimation of stroke currents measured on the interconnected LPS.

The pathogenesis of inflammatory liver diseases and development of liver fibrosis involves hepatocytes as well as non-parenchymal liver cells like resident liver macrophages (Kupffer cells (KC)), Stellate cells and endothelial cells. Kupffer cells play a critical role in liver (patho)physiology and in the defense of the liver during inflammation. They constitute about 50% of non-parenchymal cells and are the largest population of tissues macrophages in the body. Infections, toxins (lipopolysacharide (LPS)), parenchymal damage and stresses stimulate the inflammatory response of Kupffer cells with the following secretion of bioactive factors, cytotoxicity, antigen processing, etc. Resident liver macrophages are the main producers of inflammatory mediators in the liver. Among them there are prostanoids (prostaglandin (PG) E2 and thromboxane (Tx) A2), cytokines (e.g. interleukin (IL)-1,-6, -10, tumor necrosis factor (TNF) α) and inorganic mediators like nitric oxide (NO). Macrophages-derived products play opposing roles in the development of liver fibrogenesis: IL-1β, TNFα, IL-6, transforming growth factor (TGF)-β and TxA2 (pro-fibrogenic mediators) promote whereas PGE2, IL-10 and nitric oxide (anti-fibrogenic mediators) suppress liver fibrogenesis. The present study shows the production of PGE2 and TxA2 by resident liver macrophages upon prolonged activation by LPS and the characterization of biosynthesis pathways. The production of PGE2 and TxA2 is followed during 24 h after stimulation of macrophages with LPS. The involvement of enzymes is measured on the RNA level (RT-PCR), protein level (Western blot analysis) and activity (activity assays), respectively. The amounts of released prostanoids are measured at time points 2, 4, 8 and 24 h after LPS stimulation. The production of PGE2 is very low without stimulation, shows a delay within the first few hours after stimulation with LPS, and thereafter linearly increases up to 24 h. TxA2 production is very low without stimulation, and increases without a time-delay after the addition of LPS. Prostanoid biosynthesis is inhibited by dexamethasone. The present study shows the involvement and regulation of the AA cascade by the following enzymes: cPLA2: is expressed in resting Kupffer cells; cPLA2 expression and phosphorylation is increased by LPS, dexamethasone suppresses the LPS effect, localization in membrane fraction. COX-1: is expressed in resting Kupffer cells; COX-1 expression is not influenced by LPS and dexamethasone. The COX-1 inhibitor SC560 suppresses the LPS-induced production of PGE2 and TxA2 (8h and 24h), localization predominantly in membrane fraction. COX-2: is almost not expressed in resting Kupffer cells; COX-2 expression is highly increased by LPS, dexamethasone suppresses the LPS effect. The COX-2 inhibitor SC236 inhibits the production of PGE2 and TxA2 at 8h by about 77% and 20%, and at 24h by about 42% and 34%, respectively, localization predominantly in membrane fraction. mPGES-1: is almost not expressed in resting cells; mPGES-1 expression is highly increased by LPS, dexamethasone suppresses the LPS effect, localization in membrane fraction. mPGES-2: is expressed in resting Kupffer cells; mPGES-2 expression is slightly increased by LPS, localization predominantly in membrane fraction. cPGES: is expressed in resting Kupffer cells; LPS has no effect, localization predominantly in soluble fraction. TxA2 synthase: is expressed in resting Kupffer cells; LPS and dexamethasone have no effect, localization predominantly in membrane fraction. Treatment of Kupffer cells with IL-1ß and TNF-α leads to an enhanced release of PGE2 and TxA2 and upregulate the expression of cPLA2, COX-2 and mPGES-1. IL-6 has no effect on prostanoid production. In contrast, IL-10 suppresses the LPS-induced production of PGE2 and TxA2 and expression of cPLA2, COX-2 and mPGES-1. Resting Kupffer cells release very low amounts of NO and do not express iNOS, nNOS and eNOS. LPS, TNF-α and IL-1ß upregulate NO release and the expression of iNOS whereas dexamethasone and IL-10 downregulate NO release and the expression of iNOS. PGE2 suppresses the LPS-induced release of NO but enhances the cytokine-induced release of NO. NO induces a release of PGE2. Thus, the study demonstrates a crosstalk between prostanoids, nitric oxide and cytokines in Kupffer cells under inflammatory conditions and demonstrates a possible anti-fibrogenic effect of PGE2 in the process of liver fibrogenesis.

Les réactions de type hypersensibilité parfois observées au début de séances d'hémodialyse ont été corrélées à la présence, dans les bains de dialyse, d'endotoxines ou lipopolysacharides (LPS) provenant de bactéries de type gram négatif. Nous nous sommes tout d'abord attachés au développement d'un système de production de bains de dialyse stériles et apyrogènes qui soit intégrable au matériel existant. Notre étude nous a permis de rejeter les dispositifs d'ultrafiltration utilisant des membranes organiques planes ou des membranes minérales tubulaires. Nous avons proposé l'emploi d'un hémofiltre à fibres creuses en polyamide qui s'avère un bon compromis puisque, dans des conditions de contamination normales, le bain produit est stérile et apyrogène et la faible perte de charge générée par le filtre permet son intégration aux générateurs de bains existants. Nous avons également déterminé plus précisément la masse moléculaire des LPS par électrophorèse et par filtrations successives sur des membranes de seuils de coupure décroissants. Nous avons ainsi pu mettre en évidence les LPS après filtration sur des membranes de 5 kDa pour les LPS de type R. Dans une dernière étape, nous avons cherché à modifier la membrane de dialyse pour augmenter sa rejection des LPS. Un revêtement de la face dialysat de la membrane par un polymère organique polycationique nous a permis d'obtenir des absorptions notables de LPS. La conjonction des deux méthodes que nous avons proposées, ultrafiltration des bains de transfert des LPS vers le compartiment sanguin. Des essais cliniques nous permettront de valider ce procédé.

Patienten mit chronischem Nierenversagen leiden sowohl klinisch als auch subklinisch unter Entzündungsepisoden. Um einen Frühindikator der Mikroinflammation zu finden, wurde die Expression funktioneller monozytärer Oberflächenantigene (HLA-DR, CD14, CD16, TLR2 (extra-, intrazellulär), TLR4 (extra-, intrazellulär), CD80, CD86), das Zytokinexpressionsprofil (IL1, IL6, IL10, TNFa, TGFb) und der ultrastruktrurelle Phänotyp des Monozyten in vivo bzw. in vitro untersucht. Dabei wurde strikt zwischen der Membranproteinexpression auf antiinflammatorischen (CD14++CD16-), proinflammatorischen (CD14++CD16+, CD14dimCD16+) und CD14+ bzw. CD16+ Monozyten unterschieden und parallel die Serumspiegel von Parathormon (PTH), C-reaktivem Protein (CRP), Calcium und Phosphor untersucht. Inwieweit monozytäre Aktivierungsmarker zum immunologischen Monitoring geeignet sind, sollte vergleichend zwischen Gesunden, Hämodialyse-(HD)- und Peritonealdialyse-(CAPD)-Patienten untersucht werden. Zusätzlich wurde die Expression in einem in-vitro-Zellkulturmodell vergleichend betrachtet. Der Serum-PTH-Spiegel fiel nach Injektion des Vitamin D-Derivates Paricalcitol, der Serumcalciumspiegel stieg signifikant innerhalb des oberen Referenzbereiches bei Patienten mit sekundärem Hyperparathyroidismus drei Wochen nach Beginn der Therapie. Die HLA-DR, extrazelluläre TLR2, intrazelluläre TLR4, CD80, CD86 Expression fiel nach Paricalcitolinjektion. Paricalcitol erhöhte die Anzahl antiinflammatorischer und erniedrigte die Anzahl proinflammatorischer Monozyten. Beim ultrastrukturellen Vergleich zeigte sich eine deutliche Häufung von elektronendichten Granula bei Paricalcitol-inkubierten Zellen im in-vitro-Versuch. Hierbei handelt es sich mit an Sicherheit grenzender Wahrscheinlichkeit um Lysosomen, was die These einer erhöhten phagolysosomalen monozytären Aktivität unter Paricalcitol-Inkubation stützt. Sowohl bei 48- als auch bei 72-stündiger Inkubation in Primärzellkultur wirkte Paricalcitol antiinflammatorisch, indem es Aktivierungsmarker des Monozyten (HLA-DR, TLR2 (extrazellulär), TLR4 (extrazellulär)), die Anzahl proinflammatorischer Monozyten und die Synthese proinflammatorischer Zytokine (IL1, IL6, TNFa) supprimierte. Dialysepatienten unterliegen einem erhöhten Eintrag von Endotoxin (=LPS) über die Dialysemembran, das in der Regel durch Bindung an den LPS-Rezeptor (=CD14) detoxifiziert wird. Dieser wird nicht nur nach LPS Inkubation, sondern v.a. nach Paricalcitol Inkubation vermehrt exprimiert. LPS erhöhte die Anzahl der proinflammatorischen Monozyten in Zellkultur und reflektierte damit den steady state des HD-Patienten. Aktivierungsmarker von Monozyten unterschieden sich zudem bei vergleichender Betrachtung zwischen CAPD-, HD-Patienten und Gesunden und außerdem im Vergleich vor und nach HD. Die intrazelluläre TLR2 und TLR4 Expression von CAPD-Patienten und HD-Patienten war gegenüber Gesunden erniedrigt, während die CD14 Expression signifikant erhöht war. HD-Patienten zeigten einen erhöhten Anteil proinflammatorischer Monozyten vergleichend zu Gesunden aber auch zu Patienten unter CAPD-Substitutionstherapie. Unmittelbar nach HD fielen die proinflammatorischen Monozyten, während v.a. die Expression von extrazellulärem TLR2, intrazellulärem TLR2 und intrazellulärem TLR4 stieg. Somit unterliegt der HD-Patient einer stärkeren Mikroinflammation als der CAPD-Patient. CAPD-, HD- und auch Patienten mit sekundärem Hyperparathyroidismus zeigen Zeichen einer Mikroinflammation. Dabei war CRP (derzeitiger Routineparameter) kein probates diagnostisches Mittel der Entzündung bei CAPD- und HD-Patienten, des Weiteren nicht geeignet zwischen beiden Patientengruppen zu unterscheiden. Die Verteilung der monozytären Subpopulationen und Expression monozytärer Aktivierungsmarker unterschied sich hinreichend zwischen CAPD- und HD-Patienten. Paricalcitol moduliert funktionelle monozytäre Antigene und Zytokine in vivo und in vitro und wirkt damit der Mikroinflammation und dem Immundefekt des CNI-Patienten entgegen. Der Monozyt wirkt ambivalent und initiiert seine „eigene“ Gegenregulation zur Inflammation, die beim Patienten unter Nierenersatztherapie Endotoxin-vermittelt ist. Vitamin D-Derivate wie Paricalcitol wirken nicht nur auf die Calcium-Phosphat-Homöostase sondern auch immunmodulatorisch, indem sie auf monozytäre Antigene, wie Rezeptoren der angeborenen Immunabwehr, Einfluss nehmen.

El concentrat de plasma assecat per polvorització (SDP) i el d’immunoglobulines (IC) són suplements dietètics que han estat àmpliament utilitzats en l’alimentació dels animals de granja i que han estat proposats com alternativa a l’ús dels antibiòtics com a promotors del creixement, especialment durant el període posterior al deslletament. Aquests suplements incrementen la taxa de creixement degut, en part, a que atenuen la resposta del sistema immunitari. Per exemple, s’ha pogut observar que la suplementació dietètica amb proteïnes plasmàtiques redueix la sobreestimulació immunitària en un model de malaltia inflamatòria intestinal. El sistema immunitari mucosal comú connecta els llocs inductors (en aquest cas la mucosa intestinal) amb els llocs efectors (com les mucoses naso- i broncoalveolar o la genito-urinària), que facilita el moviment de cèl•lules immunitàries entre teixits allunyats anatòmicament i permet que la dieta moduli la resposta associada a infeccions extraintestinals. Així doncs, ens hem plantejat si la suplementació dietètica amb proteïnes plasmàtiques podia modular la resposta immunitària en una inflamació pulmonar aguda. Primerament es va establir una pauta adequada per obtenir un model d’inflamació pulmonar robust i reproduïble. Seguidament s’han estudiat els efectes dels suplements SDP i IC sobre la resposta immunitària en aquest model d’inflamació, en ratolins acabats de deslletar. S’han analitzat cèl•lules i mediadors inflamatoris involucrats tant en la resposta innata, com en la resposta adaptativa. Finalment, també s’han estudiat els efectes dels pinsos experimentals sobre diferents elements que intervenen en la resolució de la resposta inflamatòria, com les cèl•lules reguladores i les citocines antiinflamatòries. L’administració de LPS produeix una migració de leucòcits i un alliberament massiu de citocines i quimiocines proinflamatòries, que recluten i activen monòcits i neutròfils a l’espai alveolar i al teixit pulmonar. A més estimula la immunitat adaptativa, en la que hi ha un increment considerable de limfòcits Th activats, així com una gran producció de citocines. Ambdues dietes disminueixen el reclutament de leucòcits, sobretot els neutròfils i els monòcits. Tot i que la resposta innata és més extensa, les dietes són més efectives sobre la resposta adaptativa, on redueixen les poblacions limfocitàries estimulades per l’LPS tant a pulmó com a sang. Els suplements també atenuen de forma notable l’efecte de l’LPS sobre l’expressió de citocines i quimiocines proinflamatòries a l’espai alveolar. Els efectes de l’SDP i l’IC estan mediats per canvis en l’expressió de citocines antiinflamatòries, ja que augmenten la concentració de IL-10 a pulmó i a jejú, i la del TGF-β a pulmó. Ambdues dietes redueixen el balanç entre els limfòcits Th activats i els limfòcits T reguladors, d’aquesta manera atenuen la magnitud de la resposta inflamatòria i augmenten la concentració de mediadors essencials per la reparació del teixit. Els resultats d’aquest estudi indiquen que els suplements amb proteïnes plasmàtiques poden limitar la resposta immunitària pulmonar i podrien ser útils en la prevenció i atenuació de les malalties inflamatòries extraintestinals. / The concentrated spray-dried plasma (SDP) and immunoglobulin (IC) are dietary supplements that have been widely used in the diet of farm animals and proposed as an alternative to the use of antibiotics as growth promoters, especially in the subsequent weaning period. These supplements increase the growth rate, in part because they attenuate the response of the immune system. The common mucosal immune system connects the inductive sites (in this case the intestinal mucosa) with the effector sites (as nasopharyngeal and bronchoalveolar mucosa or genito-urinary), which facilitates the movement of immune cells between anatomically distant tissues and allows the diet to modulate the associated response to intestinal infections. Consequently, the aim of the present thesis was to evaluate whether the dietary supplementation with plasma proteins could modulate the immune response in an acute lung inflammation. First, a suitable pattern was established in order to obtain a robust and reproducible inflammatory lung model. Next, the effects of SDP and IC supplementation on the immune response in this inflammation model were studied in newly weaned mice. Cells and inflammatory mediators, involved either in innate response as in adaptive response, have been analyzed. Finally, the effects of experimental feed on different elements involved in the resolution of the inflammatory response, such as regulatory cells and inflammatory cytokines, have also been assessed. Both diets decreased the recruitment of leukocytes. Although the innate response is more extensive, the diets are more effective on the adaptive response. The supplements also significantly attenuated the effect of LPS on the expression of pro-inflammatory cytokines and chemokines in the alveolar space. The effects of SDP and IC are mediated by changes in the expression of anti-inflammatory cytokines. Both diets reduced the balance between activated Th cells and regulatory T cells, in such a way that, the magnitude of the inflammatory response is attenuated and the concentration of essential mediators for tissue reparation is increased. In conclusion, the results of this study indicate that plasma protein supplementation is able to limit the pulmonary immune response and could be useful in the prevention and attenuation of the extraintestinal inflammatory diseases.