Poor egg quality is the leading cause of pregnancy loss and Down's syndrome. While even eggs in young women frequently contain an incorrect number of chromosomes and are therefore unlikely to give rise to a viable pregnancy, the incidence of chromosomally abnormal eggs increases strikingly with advancing maternal age. Why egg quality declines dramatically as women approach their forties remains one of the outstanding questions in developmental biology. This PhD thesis demonstrates how unforeseen features of kinetochore organization that are unique to meiosis render this cell division process in mammals particularly prone to errors. Firstly, my results uncovered an unexpected multi-subunit organization of the meiotic kinetochore, which is widely conserved across mammals and biases eggs towards errors. Secondly, I identified two independent mechanisms that predispose eggs from older women to aneuploidy. The first mechanism affects the fidelity of meiosis I. My analysis revealed that human oocytes challenge the paradigm that sister kinetochores are fully fused. Instead, I demonstrated that sister kinetochores disjoin as women get older, which promoted erroneous kinetochore-microtubule attachments. This in turn allowed chromosomes to rotate on the spindle and provided a mechanistic explanation for reverse segregation - a recently discovered meiotic error that is unique to humans. Secondly, I pioneered the use of super-resolution microscopy to study chromosome architecture in human eggs and discovered that individual kinetochores during meiosis II in mammals are composed of previously unidentified subdomains. In young females, these subdomains are joined together by cohesin complexes. With age, kinetochores fragment into two pieces. Fragmented kinetochores frequently attach merotelically to spindle microtubules, which predisposes aged eggs to errors. What severely hinders our progress in identifying causes of human infertility is that numerous features of human meiosis are not represented in mice. To overcome this challenge, I developed an experimental platform to mimic the age-related changes that occur in humans in oocytes from young mice. I achieved this by extending the applications of Trim-Away, a novel method to degrade endogenous proteins even in primary cells, to partially deplete proteins. Furthermore, I established a new experimental model system to study human-like aspects of meiosis in live non-rodent cells in real time: pig oocytes. Together, these results set foundations for new therapeutic approaches to extend reproductive lifespan by counteracting the age-related loss in kinetochore integrity that this study identified. Furthermore, partial Trim-Away and studying meiosis in pigs opens new directions for meiotic research.

Every fall, Eduardo Zayas-Bazán, a native of Cuba who was a veteran of the 1961 Bay of Pigs invasion and who taught Spanish at East Tennessee State University for over 30 years, hosts a Cuban-style pig roast in Tennessee for family and friends. "Among my fondest memories are the pig roasts we had in Cuba on special occasions," said Zayas-Bazán, who marinates his pigs in grapefruit juice mixed with garlic and oregano, and cooks them in a contraption that he says "looks like a shoe box with a grill inside."" With the annual pig roast as context, the film tells the story of Zayas-Bazán's life, both in Cuba and the U.S. / https://dc.etsu.edu/etsu_books/1098/thumbnail.jpg

Cystic fibrosis (CF) is the most common autosomal recessive genetic disease in Caucasian populations. CF affects multiple organ systems including pancreas, liver, intestines, sweat glands, and male reproductive organs, however the leading cause of morbidity and mortality in CF patients is chronic lung disease. CF is caused by a mutant cystic fibrosis transmembrane conductance regulator (CFTR) gene which leads to chloride (Cl-) and bicarbonate (HCO3-) anion dysregulation at the airway surface. Without adequate anion exchange, thick, viscous mucus accumulates at the airway surface allowing bacterial colonization to occur. Complementing CFTR in the appropriate airway cells restores the anion channel activity in CFTR-deficient cells. The ultimate goal for CF gene therapy is to design an integrating vector that would lead to persistent and efficient expression of CFTR in the airways. Performing gene therapy experiments is dependent upon a relevant animal model. The CF pig is a large animal model similar in size, anatomy, and physiology to humans. Importantly, the CF pig recapitulates human lung disease. From the CF pig, we have learned much about CF lung disease and have developed relevant assays to measure anion channel correction. We have learned that loss of CFTR leads to a decreased airway surface ASL pH, bacterial killing ability, and increased mucus viscosity. Standardized assays have been developed to evaluate the change in current by Ussing chambers, ASL pH, bacterial killing in vivo and ASL pH and viscosity on primary airway cultures in vitro. Ultimately, these metrics allow us to make conclusions about the efficiency of CFTR restoration. Viral vectors are promising candidates for CF gene therapy. Viral vectors such as adenovirus (Ad), adeno-associated virus (AAV), and pseudotyped lentiviral vectors such as feline immunodeficiency virus (FIV) or human immunodeficiency virus (HIV) can efficiently transduce airway cells and express CFTR. Ad and AAV have both been tested in CF clinical trials, but CFTR expression was transient, if detected at all. Understanding vector biology and overcoming barriers in the lung have allowed us to improve vector delivery to the airways. However, the next major hurdle was achieving persistent expression. Ad and AAV are both transiently expressing vectors, and vector readministration is implausible due to the presence of neutralizing antibodies that develop against the vector. Creating a hybrid nonviral/viral vector in which the integrating nonviral piggyBac transposon system is delivered by an Ad or AAV vector has allowed us to achieve persistent expression in mice. In a third integrating vector system, lentiviral vectors have historically been challenging to work with due to low titer levels. However, improvement in vector purification methods have allowed us to validate a lentiviral vector as a viable gene therapy option. In total, we have validated three integrating vector systems by restoring CFTR to CF pigs to correct the phenotypic defect.

Cancer is the second deadliest disease in the United States with an estimated 1.69 million new cases in 2017. Medical imaging modalities, such as computed tomography (CT) and magnetic resonance imaging (MRI), are widely used in clinical medicine to detect, diagnose, plan treatment, and monitor tumors within the body. Advances in imaging research related to cancer assessment have largely relied on consented human patients, often including varied populations and treatments. Tumor bearing mouse models have been highly valued for basic science research, but imaging focused applications are limited by the translational ability of micro imaging systems. Pig models are well suited to bridge the gap between human cohorts and mouse models due to similar anatomy, physiology, life-span, and size between pigs and humans. These models provide the opportunity to advance medical imaging while simultaneously characterizing progressive changes resulting from an intervention, exposure, or genetic modification. We present a foundation for effectively characterizing disease models in pigs, susceptible to tumor development, using longitudinal medical image acquisition and post-processing techniques for quantification of disease. Longitudinal, whole-body protocols were developed with CT and MRI. Focus was placed on systematic process, including transportation, anesthesia and positioning, imaging, and environmental controls. Demonstration of the methodology was achieved with six pigs (30-85 kg) with four to seven imaging time points acquired per animal. Consistent positioning across time points (CT to CT) and within time points (CT to MRI) was assessed with distance measures obtained from the skeleton following rigid registration between images. Alignment across time points was achieved with an average value of 16.51 (± 12.46) mm observed all acquired measurements. For consistent, retrievable, and complete qualitative assessment of acquired images, structured reports were developed, including assessment of imaging quality and emphasis on tumor development throughout the body. Reports were used to perform a systematic, semi-qualitative comparison of CT and MRI lung assessment with an overall agreement of 72% in detection of disease indicators. A multi-level registration algorithm was developed to align anatomic structures of interest in the acquired longitudinal datasets. The algorithm consisted of initialization followed by repeated application of a core registration framework as the input data reduced in image field of view. It was applied to align regions of interest in the brain, upper right lung, and right kidney. Validation was performed with overlap (range = [0.0,1.0], complete overlap = 1) and distance measures (range = [0.0, ∞], perfect match = 0.0) of corresponding segmentations with overall results of 0.85 (± 0.11) and 0.41 (± 0.83) mm, respectively. An extension of the algorithm was created, demonstrating the ability to incorporate directional growth and feature extraction measurements into longitudinal tumor progression monitoring. Techniques were applied to a phantom dataset showing solid tumor growth and transition from a non-solid to part-solid lesion in the lungs. Finally, the developed methods – imaging, structured reporting, registration, and longitudinal feature extraction – were applied to four different porcine models pre-disposed to tumor development. 1) A genetically modified Li-Fraumeni (TP53R167H/+/TP53R167H/R167H) background model showing the development of osteosarcoma and lymphoma. 2) A TP53R167H/+ animal with exposure to crystalline silica showing progression of silicosis in the lungs. 3) TP53R167H/+/TP53R167H/R167H animals with exposure to radiation for targeted sarcoma development and 4) TP53R167H/+ pigs with conditional KRASG12D/+ mutation activated in the lung and pancreas. Whole-body and targeted imaging protocols were developed for each model and qualitatively interpreted by a radiologist using structured reports. Multi-level registration was used to align identified tumors and longitudinal features were extracted to quantitatively track change over time. Overall, the developed methods aided in the effective, non-invasive characterization of these animals.

The initial focus of this PhD project was on comparative gene mapping. Comparative gene mapping is facilitated by consensus PCR primers which amplify homologous gene fragments in many species. As a part of an international co-ordinated programme of comparative mapping in pigs, 47 CATS (Comparative Anchor Tagged Sequence) consensus primer pairs for loci located on human chromosomes 9, 10, 20, and 22, were used for amplifying homologous loci in pigs. After optimization of PCR conditions, 23 CATS products have confirmed by comparison with homologous sequences in GenBank. A French somatic cell hybrid panel was used to physically map the 6 porcine CATS products distinguishable from rodent background product, namely ADRA1A, ADRA2A, ARSA, GNAS1, OXT and TOP1. Of these, the map location of ADRA1A and OXT showed inconsistency with the previously recognised conserved relationship between human and pig. The other four loci mapped to positions consistent with known syntenic relationships. Despite low levels of polymorphism, frequently indistinguishable rodent and porcine products in somatic hybrids and some confusion of identity of gene family members, these CATS primers have made a useful contribution to the porcine-human comparative map. The focus of the project then changed to genetic and molecular characterisation of endogenous retroviruses in pigs and their relatives. Pigs are regarded as a potentially good source of organs and tissues for transplantation into humans. However, porcine endogenous retroviruses have emerged as a possible problem as they can infect cultured human cells. Two main types of pig retrovirus, determined by envelope protein, PERV-A and PERV-B, are widely distributed in different pig breeds and a third less common type, PERV-C, has also been recognised. Endogenous retroviruses were analyzed from the Westran (Westmead transplantation) inbred line of pig, specially bred for biomedical research. Thirty-one 1.8 kb env PCR product clones were sequenced after preliminary screening with the restriction enzymes KpnI and MboI. Five recombinant clones between A and B were identified. 55% of clones (17/31) sequenced had stop codons within the envelope protein-encoding region, which would prevent the retrovirus from making full-length envelope protein recognizable by cell-surface receptors of the virus. The endogenous viruses were physically mapped in Westran pigs by FISH (Fluorescence In Situ Hybridisation) using PERV-A and PERV-B envelope clones as probes. Preliminary FISH data suggest that there are at least 22 PERVs (13 PERV-A and 9 PERV-B) and the chromosomal locations of these in the Westran strain are quite different from European Large White pigs. The sequences and mapping results of inbred Westran pig suggest that there are relatively few PERV integration sites compared with commercial pigs and further that a large proportion of clones are defective due to premature stop codons in the envelope gene. To investigate the relationship of endogenous retroviruses in peccaries and pigs, a set of degenerate primers was used to amplify peccary retroviral sequences. The sequences of two putative retroviral clones showed close homology, albeit with a 534 bp deletion, to mouse and pig retroviral sequences. Also, four non-target sequences were amplified from peccary with the degenerate retroviral primers. They are a part of the peccary cofilin gene, a SINE, and a sequence containing a microsatellite. The peccary endogenous retroviral sequences are significant in that they are the first such sequences reported in peccary species and repudiate old claims in the literature that peccaries do not have C-type retroviral sequences.

Immunological stressors, in the form of clinical and sub-clinical disease are currently controlled using both prophylactic antibiotics in-feed, and therapeutic antibiotic treatment. Respiratory disease, primarily Actinobacillus pleuropneumoniae (App) infection, is recognised as a major factor causing reduced productivity in pigs. This thesis reports investigations into the use of novel immunomodulators in particular Interleukin 4 (IL-4) and Interleukin 1 receptor antagonist (IL-1ra) as alternatives to antibiotics to treat App infection. Immunological and molecular biological assays were used to investigate and accumulate data. An in vitro study undertaken to find potential anti-inflammatory substances, revealed that Interleukin 8 (IL-8) mRNA production stimulated by PMA or LPS in whole pigs' blood was suppressed by IL-4. IL-1ra also suppressed stimulated IL-8 mRNA production by heat killed App bacteria (KB) in vitro. An acute LPS challenge in pigs in vivo however, showed no variation in illness or weight loss between pigs treated prophylactically with anti-inflammatory substance (IL-4 and IL-1ra) and saline treated pigs. The use of plasmids as a delivery system for anti-inflammatory substance did not show promise since it did not enhance growth or prolong the expression of the substances in the pigs. However, in the chronic App challenge model IL-4 and IL-1ra administered prophylactically in vivo showed an ability to improve growth. The therapeutic administration of IL-4 and IL-1ra to App challenged pigs showed no difference in pigs' growth, regardless of the treatment or control administered. To conclude, IL-4 and IL-1ra showed promise when administered prophylactically and improved growth and abrogated disease under conditions of App challenge. However when IL-4 and IL-1ra where administered therapeutically they did not perform as well. Moreover these compounds have potential as a commercial application to reduce the growth reduction caused by disease such as App.

<p>Septic shock is frequently seen in intensive care units and is associated with significant mortality. Endotoxin – a major mediator of the pathophysiologic responses – is released during lysis of Gram-negative bacteria. These responses can be mimicked in the endotoxaemic pig.</p><p>This thesis focuses on the following topics: the inflammatory and pathophysiological responses to various endotoxin doses and infusion patterns; covariations between endotoxin induced inflammatory and pathophysiological responses; whether the biological effects of endotoxin can be modulated by clopidogrel and whether tobramycin or ceftazidime reduce plasma cytokine levels.</p><p>Endotoxin induced linear log-log cytokine and F2-isoprostane responses. Leukocyte and platelet responses, pulmonary compliance, circulatory variables as well as indicators of plasma leakage and hypoperfusion exhibited log-linear responses to the endotoxin dose. Biological responses to endotoxaemia such as inflammation, hypotension, hypoperfusion and organ dysfunction were more expressed when the organism was exposed to endotoxin at a higher rate. These results may facilitate the possibility to choose relevant endotoxin administration, when experiments are set up in order to evaluate certain responses to endotoxaemia.</p><p>Correlation studies between cytokines, leukocytes, platelets and the endotoxin dose were in agreement with the well-known ability of endotoxin to induce cytokine expression and to activate both primary haemostasis and leukocytes. Free radical mediated lipid peroxidation and COX-mediated inflammation correlated to cytokine expression and organ dysfunction in endotoxaemic shock. </p><p>Endotoxaemic pigs pretreated with clopidogrel, exhibited a trend towards less expressed deterioration of renal function, although blocking of ADP-induced primary haemostasis is not a key mediator of endotoxin induced deterioration of renal function.</p><p>Tobramycin did not neutralise the biological effects of endotoxin or the plasma levels of endotoxin, suggesting that these antibiotics do not bind to endotoxin.</p><p>Reduction in IL-6 was greater in pigs treated with ceftazidime and tobramycin as compared with those given saline, indicating a possible anti-inflammatory effect of both antibiotics.</p>

Pig follicle development is a complex but poorly understood process involving both gonadotrophins and local ovarian factors. A series of studies sought to investigate these intrafollicular cell-to-cell interactions. Microarray analysis combined with gene ontology revealed that the oocyte, granulosa and theca cells each expressed more than 650 potential secreted factors and receptors, including members of the TGF-β, IGF1, EGF and FGF families. Using a well-defined in vivo experimental paradigm that generates follicles and oocytes of different quality, the temporal expression of several growth factors in the oocyte, granulosa and theca cells collected from sows during the recruitment and mid-selection phases, as well as the final selection of the preovulatory follicle population before and after the LH surge was studied. IGF1 expression patterns indicated its potential for modulating granulosa and theca cell function during the selection stages, and an involvement in creating differences in follicular quality between the first and second preovulatory wave post-weaning. Transient up-regulation of AREG and EREG mRNA around the LH surge, suggested their involvement in ovulation. Results of a second study investigating TGF-β superfamily expression, suggested a role for GDF9 in follicle selection through the up-regulation of TGFBR1 expression, while BMP15 could be involved in ovulation through the up-regulation of BMPR1B. Expression of angiogenesis-related genes during follicle development was also investigated. During mid-selection, ANGPT2 may allow VEGFA and similar factors to stimulate vascular development or destabilize the vasculature and cause follicular atresia, while ANGPT1 may be required in the preovulatory follicle population. Associations among the transcription factor HIF1A, VEGFA and ANGPT1, suggested interactions between the ligands in regulation of angiogenesis. Finally, the effects of the pig oocyte on cumulus cell function was assessed by co-culturing cumulus cell complexes with or without denuded oocytes isolated from large oestrogenic follicles. Presence of oocytes decreased FSHR and increased HSD3B expression, potentially stimulating progesterone while attenuating oestradiol production. In conclusion, oocytes were shown to control cumulus cell function in a way that reflects their specific environment and further evidence was obtained for a complex network of growth factors and receptors in the follicle and their involvement in regulation of pig follicle development. / Animal Science

Recent advances in genomics now allow for the identification of the genes and mutations that underlie the heritability of agronomically important traits in livestock. The corresponding genes are said to map to Quantitative Trait Loci (QTL), and the mutations referred to as Quantitative Trait Nucleotides (QTN). The most commonly used approach relies on positional cloning which typically proceeds in three steps: QTL mapping by linkage analysis, QTL fine-mapping by linkage disequilibrium or association analysis, and QTN identification combining haplotype analysis and functional assays. Knowledge of QTL and QTN provides insights into the genetic architecture of complex traits and physiology of production traits, and opens novel possibilities for enhanced selection referred to as Marker Assisted Selection (MAS). This thesis is devoted to QTN identification of a QTL that was previously mapped to pig chromosome 2 and fine-mapped to a 250 Kb segment encompassing the imprinted IGF2 gene. The QTL was shown to have a major post-natal effect on muscle mass and fat deposition, and to be subject to parental imprinting as only the padumnal chromosome affects the phenotype. To identify the QTN we have first generated 32 Kb and 56 Kb of finished porcine sequence encompassing the IGF2 and H19 genes, respectively. The corresponding sequences were annotated including definition of gene models, identification of interspersed repeats and determination of 97 sequence elements that are highly conserved between pig, human and mouse. We have then resequenced 28 Kb encompassing the IGF2 gene for 15 boar chromosomes for which the QTL genotype had been determined by progeny-testing or Marker Assisted Segregation Analysis (MASA). This revealed 258 polymorphisms of which only one (Int3-3072G>A) cosegregated perfectly with QTL genotype. The corresponding single nucleotide polymorphism (SNP) is a G to A transition affecting one of the highly conserved sequence elements located just downstream of differentially methylated region 1 in intron 3. We have demonstrated that the Int3-3072 A allele associated with increased muscle mass is also associated with increased IGF2 mRNA levels in post-natal striated muscle (but not in pre-natal muscle nor in pre- and post-natal liver). However, the Int3-3072G>A SNP does not alter imprinting nor allele-specific methylation. Using a luciferase reporter assay, we then demonstrated that the Int3-3072 A allele reduces the cis activity of a silencer element, and using an electrophoretic mobility shift assay (EMSA), that it abrogates binding of a nuclear factor assumed to be a trans-acting silencing factor. Taken together both genetic and functional evidence strongly support the conclusion that the Int3-3072G>A SNP is the causative SNP. The thesis is concluded by a discussion that (i) highlights the factors that make domestic animals a unique resource for the molecular dissection of complex phenotypes, (ii) comments the Asian origin of the Int3-3072A allele associated with increased muscle mass, (iii) describes recent advances in characterizing the trans-acting silencing factor binding to the Int3-3072G allele, (iv) pinpoints statistical issues related to the detection of imprinted QTL, (v) reports on the utility of the Int3-3072G>A SNP for MAS applied to pig breeding, and (vi) makes projections on how latest progress in genome analysis will affect positional identification of QTN in the near future. Grâce aux progrès récents en génomique, il est maintenant possible didentifier les gènes et mutations qui sous-tendent lhéritabilité des caractères de production chez les animaux de rente. Ces gènes se localisent au niveau de Loci de Traits Quantitatifs (QTL), et les mutations correspondantes sont qualifiées de Nucléotides de Traits Quantitatifs (QTN). La démarche expérimentale la plus couramment utilisée est le clonage positionnel. Celui-ci comprend trois étapes: cartographie de QTL par analyses de liaison génétique, cartographie fine de QTL par études dassociation exploitant le déséquilibre de liaison, et identification de QTN par combinaison danalyses haplotypiques et fonctionnelles. Lidentification de QTL et QTN non seulement révèle larchitecture génétique des phénotypes complexes que sont les caractères de production, ainsi que les rouages moléculaires qui les sous-tendent, mais ouvre également des possibilités nouvelles de sélection plus performante dite Assistée par Marqueurs (MAS). Cette thèse est consacrée à lidentification dun QTN correspondant à un QTL dabord localisé sur le chromosome 2 du porc, et ensuite cartographié finement dans un segment chromosomique de 250 Kb comprenant le gène IGF2. Le QTL en question a un effet post-natal majeur sur la croissance musculaire et le dépôt graisseux. Il est soumis à lempreinte parentale, lallèle paternel étant le seul à influencer le phénotype. Afin didentifier le QTN, nous avons tout dabord généré 32 Kb et 56 Kb de séquences finies, comprenant respectivement les gènes IGF2 et H19. Les séquences correspondantes ont été annotées bioinformatiquement, y compris la définition de modèles gèniques, lidentification de séquences répétées dispersées, ainsi que de 97 éléments de séquence fortement conservés chez le porc, lhomme et la souris. Nous avons ensuite re-séquencé 28 Kb chevauchant le gène IGF2 pour 15 chromosomes dont le génotype au niveau du QTL fut préalablement déterminé par testage de descendance ou Ségrégation Assistée par Marqueurs. Cet exercice a révélé 258 polymorphismes dont un seulement (Int3-3072G>A) correspondait parfaitement aux génotypes QTL. Ce polymorphisme est une transition G à A affectant un des 97 éléments hautement conservés, situé juste en aval de la région différentiellement méthylée (DMR1) dans lintron 3. Nous avons ensuite démontré que lallèle Int3-3072 A, associé à une augmentation de la masse musculaire, est également associé à une augmentation des taux dARNm IGF2 dans le muscle strié post-natal (mais non dans le muscle strié pré-natal, ni dans le foie pré- et post-natal). Par contre, le polymorphisme Int3-3072G>A naffecte ni état dempreinte ni de méthylation allèle-spécifique du gène. Nous avons ensuite démontré à laide dun test rapporteur de type luciférase que lallèle Int3-3072 A réduit lactivité dun élément silenceur agissant en cis, et à laide dun test de type EMSA quil empêche la liaison dun facteur nucléaire. Conjointement, ces données génétiques et fonctionnelles démontrent que le polymorphisme Int3-3072G>A SNP correspond bien au QTN. Nous concluons la thèse par une discussion dans laquelle nous (i) démontrons pourquoi les animaux domestiques offrent des possibilités uniques pour la dissection moléculaire de phénotypes complexes, (ii) commentons lorigine asiatique de lallèle Int3-3072A associé à une augmentation du développement musculaire, (iii) décrivons les progrès récent dans lidentification du facteur nucléaire reconnaissant spécifiquement lallèle Int3-3072G, (iv) attirons lattention sur les artéfacts statistiques associés à la détection de QTL soumis à lempreinte, et (v) discutons limpact de nouvelles technologies génomiques sur le clonage positionnel de gènes.

Sepsis is a common diagnose in the intensive care population, burdened with a high mortality. The systemic inflammatory reaction underlying the development of septic organ dysfunction can be modeled using Gram-negative bacterial lipopolysaccharide, endotoxin. This thesis used a porcine endotoxemic experimental sepsis model to address clinical questions difficult to answer in clinical trials; furthermore a model of secondary sepsis was developed. No additional effect on the development of renal dysfunction by tobramycin was found, indicating that a single dose of tobramycin does not further compromise renal function in inflammatory-induced acute kidney injury. Antiendotoxin treatment had no measurable effect on TNF-α-mediated toxicity once the inflammatory cascade was activated. There was an effect on the leukocyte response that was associated with improvements in respiratory function and microcirculation, making it impossible to rule out fully the beneficial effect of this strategy. However, the effects were limited in relation to the magnitude of the endotoxin concentration reduction and the very early application of the antiendotoxin measure. The lungs stood out compared to the other organ systems as having a threshold endotoxin dose for the protective effect of endotoxin tolerance. As to the development of circulatory and renal dysfunction, tolerance to endotoxin was evident regardless of the endotoxin pre-exposure and challenge dose. There was a temporal variation of endotoxin tolerance that did not follow changes in plasma TNF-α concentrations and maximal tolerance was seen very early in the course. More pronounced endotoxin tolerance at the time of maximum tolerance was associated with a more marked hyperdynamic circulation, reduced oxygen consumption and thrombocytopenia eighteen hours later. It might be of interest to use the experimental model of long-term endotoxemia followed by a second hit, which has been designed to resemble an intensive care setting, for the study of treatment effects of immunomodulating therapies in secondary sepsis. / Paper 3, previous title as submitted: "Compartmentalization of organ endotoxin tolerance in a porcine model of secondary sepsis"