Thesis (M. Res. (Med.))--University of Hong Kong, 2006.

Pregnancy associated plasma protein A (PAPP-A) is produced in increasing amounts with advancing pregnancy, a major source being the placenta. It was originally described as a homotetramer with ~ 200,000 M[r] subunits. An alternative structure has since been proposed of a disulphide bridged complex consisting of equimolar amounts of the PAPP-A subunit and the proform of eosinophil major basic protein (pro MBP).The increasing levels of PAPP-A produced during pregnancy and reports of possible proteinase inhibitory activity and immunomodulatory activity suggested that it may be an antiarthritic agent, involved in the amelioration of rheumatoid arthritis (RA) observed during pregnancy. PAPP-A has been purified from late pregnancy plasma using ammonium sulphate precipitation, gel filtration chromatography, ion exchange chromatography and affinity chromatography. The purity has been confirmed by SDS PAGE and western blot analysis. The purified PAPP-A has been used to investigate functions of PAPP-A which may support or refute a role in the amelioration of the symptoms of RA during pregnancy. Potential immunomodulatory actions of PAPP-A may be mediated by the inhibition of cytokine production or action. The effects of PAPP-A on the production of proinflammatory cytokines and also on the production of prostaglandin E[2] (PGE[2]), an inflammatory mediator, were studied using the THP 1 human monocytic cell line and the MG-63 human osteoblast- like cell line. PGE[2] was measured by radioimmunoassay and the cytokines IL-1, IL-6 and TNFalpha using commercial ELISA kits from Biosource International. IL-1alpha stimulated the production of PGE[2] and IL-6 in the MG-63 cells, whereas PAPP-A had no significant effect on basal or IL-1alpha stimulated IL-6 and PGE[2] production. In THP 1 cells, LPS, as expected, stimulated the production of PGE[2], IL-l?, IL-6 and TNFalpha. PAPP-A alone also significantly stimulated their production at higher concentrations, having a much greater effect at higher concentrations than LPS alone. PAPP-A, when incubated with LPS, produced a greater than additive effect on the production of PGE[2], but in combination with LPS had no significant effect on the levels of IL-6 obtained with LPS alone. Binding of PAPP-A to IL-1alpha and TGFbeta was studied using [125]I-cytokines. Formation of PAPP-A cytokine complexes was determined using either native SDS PAGE or gel filtration chromatography. No evidence of cytokine binding was obtained. The binding of PAPP-A to human cartilage was also investigated, since this may facilitate any protective effects of PAPP-A, such as proteinase inhibition, on cartilage. Cartilage binding was studied using immunohistochemical methods. The results indicated that PAPP-A does bind to cartilage, suggesting a possible protective role. The effect of PAPP-A on retinoic acid stimulated resorption of bovine nasal cartilage was studied and cartilage proteoglycan release measured as glycosaminoglycans using the dimethylemethylene blue assay. PAPP-A appeared to stimulate the resorption of cartilage. A key issue was whether PAPP-A functions as a proteinase inhibitor thus preventing connective tissue breakdown. Inhibition of elastase, cathepsin B, plasmin and trypsin was studied using colorimetric and fluorescent peptide substrates. PAPP-A was shown to competitively inhibit elastase, possibly due to the electrostatic interactions between the negatively charged PAPP-A and positively charged elastase. It did not inhibit the other enzymes studied,A monoclonal antibody to PAPP-A was produced by immunization of mice with the purified PAPP-A. This is currently been evaluated as to its potential use in a screening ELISA assay for monitoring PAPP-A levels. Overall the effects of PAPP-A on monocytic cell cytokine and PGE2 production and its stimulation of cartilage proteoglycan breakdown suggest it does not have a major role in the remission of RA during pregnancy. However this work has demonstrated functions of PAPP-A not previously reported and confirmed earlier observations of inhibitory activity towards elastase. Further work is required to determine the key functions of PAPP-A during pregnancy.

Dexamethasone, a glucocorticoid, is used extensively in clinical medicine in the treatment of respiratory diseases, notably asthma. This medical effect is probably due in part to a down-regulation of many cytokines, e.g. IL-8. However, the side effects of steroids often may outweigh the beneficial effects, especially if the disease in question is of a chronic nature. This combined with steroid resistance has led to an increase in the use of non-steroidal anti-inflammatory drugs. These tend to be aspirin derivatives, and many have side-effects. I have investigated a factor that is naturally produced by the body which may alleviate the symptoms of inflammation without many of the side- effects, with an aim to providing a viable alternative. A novel steroid induced monocyte derived factor was described twenty years ago by Stevenson as part of an MD thesis submitted to this university. This remains an unusual phenomenon, as most of the effects of steroids are due to a down-regulation of release; here is one of the few examples of the reverse. Although it raised many interesting points about dispersive motility of populations of polymorphonuclear leukocytes (PMN) it did not look at individual cells, nor at many of the interesting properties of neutrophils. One of the known effects of some steroids (though not Dexamethasone), and many NSAIDs, is a reduction of adhesion of PMN to venous endothelium. Human umbilical vein endothelial cells (and bovine aorta endothelium) were used as an ex vivo model for this, and my results show there is a marked decrease in this adhesion. This effect was observed not only on resting endothelium, but also when the endothelial cells had been pre-treated with ILip or thrombin. This decreased adhesion was due to an interaction with the PMN and the factor, as shown by adhesion being reduced when protein coated coverslips were used as the substrate for adhesion. It is suspected that the reasons for this may be due to an inactivation of integrins such as Mac-1, although a role for selectins cannot be ruled out. Recent work by Diaz-Gonzalez suggests that some NSAIDs act by inducing PMN to shed L-selectin. Other effects of this steroid induced monocyte derived factor on human PMN were determined using biological and biochemical techniques. Previous work has shown a novel dispersive effect of this factor on PMN when used in a uniform concentration, and so I decided to look at the moiphology of treated PMN. Using scanning electron microscopy I observed a polarisation of the PMN, but without any raffling of the membrane, a feature that is normally observed with polarisation. This morphology was not observed with control supernatants taken from monocytes cultured in the absence of Dexamethasone. This moiphology was conserved using cells in suspension and adhered to bovine aorta endothelium. The underlying actin cytoskeleton was examined using confocal microscopy, and the microfilamentous airay was noted as being devoid of spikes, observed with activation, for example with fMLP. Late cytoskeletal controlled effects, the release of granule contents, were also investigated, and it was noted that the release of primary granule contents could be inhibited by this factor in a dose dependent manner. This fusion of granules with the plasma membrane is controlled by the activation of numerous tyrosine kinases, and follows a strict order. Secondary granule release was shown to be inhibited also, as assayed by the cleavage of type I collagen, and analysis of SDS gels. The effects on the metabolic burst showed conflicting results with inhibition being present, again in a dose dependent manner, but much of this activity was removed with increasing purification, leaving only very slight inhibition.

Endothelial cells form the inner lining of blood and lymphatic vessels. In mice only tumours of the blood vessel endothelium (haemangiomas) have been thus far reported. In the first part of this thesis is described a highly reproducible method for the induction of benign tumours of the lymphatic endothelial cells (lymphangioma) in mice, by intraperitoneal injection of incomplete Freund's adjuvant. Different criteria have been used in order to establish the nature of the induced lesion. Morphological and histophatological studies of the tumour developed in the peritoneal cavity revealed the presence of cells at various levels of vascular development. Expression of the endothelial markers PECAM/CD31, ICAM-l/CD54, ICAM 2/CDI02 as well as the vascular endothelial growth factor (VEGF) receptor Flk-I, the endothelial cell specific receptors Tie-I, Tie-2, and the lymphatic endothelial specific Flt-4 was identified. When the lesion was induced in ~- galactosidase knock-in Flt-4 +/- mice, the tumour endothelia could be stained blue in a number of tumour cells. Tumour-derived cells were propagated in vitro where they spontaneously differentiated, forming vessel-like structures. This evidence leads to the conclusion that this is the first experimental protocol for the induction of a lymphatic endothelium hyperplasia in mice peritoneum. The second part of this thesis describes the use of this model system to investigate the profile of chemokine expression in murine lymphangiomas and in lymphangioma-derived lymphatic endothelial primary cultures. Chemokines are a superfamily of small, secreted chemoattractant molecules that plays a key role in the immune cell trafficking. Although production of chemokines by vascular endothelial cells has been extensively documented, there is much less information regarding the lymphatic endothelium. The reported results are the first detailed analysis of chemokine production by lymphatic endothelial cells. Chemokines belonging to all three subfamilies (CXC, CC and C), were found to be expressed in lymphangioma. Among these molecules is remarkable the identification of CIO, a molecule previously identified only in the bone marrow. The molecular as well as functional assays performed provide an indication of the signals that mediate the recruitment of leukocytes into lymphatic vessels.

CURRENTLY UNDER EMBARGO UNTIL THE 26/4/2019: Type 2 diabetes is a metabolic disorder characterised by inflammation, insulin resistance and the inability of pancreatic β-cells to secrete enough insulin to produce a physiological effect. Obesity and high levels of triacylglycerol’s are associated with the development of Type 2 diabetes. Adipose tissue is an active endocrine organ that secretes various protein and peptide hormones, known as adipokines, which mediate important metabolic functions. In an insulin resistant and hyperglycaemic state, levels of anti-inflammatory adipokines, adiponectin, are reduced, whereas levels of pro-inflammatory cytokines, interleukin-6 and interleukin-1β, are elevated; this results in a shift from an anti- to a pro-inflammatory state that is accompanied by dysfunction and apoptosis of the pancreatic β-cells. Cannabis sativa L. has been traditionally used as an anti-inflammatory agent in Southern Africa, specifically treating snakebites, fever and malaria. Δ9-tetrahydrocannabinol is the main psychoactive compound derived from C. sativa, whereas the other major cannabinoids, cannabinol and cannabidiol, have shown anti-inflammatory and sedative properties respectively. Marrubiin is a compound derived from the plant Leonotis leonurus L. and has been traditionally used as an anti-inflammatory and anti-diabetic agent. To determine the effects of these compounds in a hyperglycaemic state, pre- and differentiated mouse adipocytes (3T3-L1 cells) were exposed for seven and fourteen days to the following treatments: Δ9-tetrahydrocannabinol, cannabidiol, cannabinol, marrubiin, anandamide (an endogenous endocannabinoid) and cannabis extract, individually and in combination, under normal glucose and hyperglycaemic conditions. Levels of adiponectin, interleukin-6, leptin, tumour-necrosis factor-α and interleukin-1β were quantified using mouse enzyme-linked immunosorbent assay kits and Oil Red O staining was carried out to determine lipid distribution and lipid droplet characteristics. Results indicate that various cannabinoids, in combination, mediate an anti-inflammatory effect by decreasing the expression of various pro-inflammatory cytokines, which may have allowed for a shift from a pro- to an anti-inflammatory state by these compounds, and may also contribute to the reduction of lipid, which may be used as a supplementary option to current diabetic treatment regimes.

Orientadores: Helena Zerlotti Wolf Grotto, Carmen Silvia Passos Lima / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T13:20:08Z (GMT). No. of bitstreams: 1 Jacober_MicheleLealVieira_M.pdf: 5411345 bytes, checksum: 3d65908f63a20305387ec02808acdd0e (MD5) Previous issue date: 2007 / Resumo: Os pacientes com câncer podem apresentar diversas anormalidades hematológicas, sendo a anemia a alteração mais comum. A etiologia da anemia é multifatorial, envolvendo mecanismos que podem estar relacionados ou não à doença (perda de sangue, hemólise, infiltração medular), ao tipo e estágio do tumor e também à terapêutica utilizada (radioterapia e quimioterapia). A redução nas taxas de hemoglobina pode estar relacionada a alterações na produção e na resposta dos precursores eritróides à Eritropoetina, à liberação de citocinas nas diversas fases da doença e também ao déficit nutricional muitas vezes encontrado nesses indivíduos. O objetivo desse estudo foi avaliar aspectos relacionados à atividade inflamatória sobre o metabolismo do ferro e a eritropoiese em pacientes com tumores sólidos. Foram coletadas amostras de sangue de 402 pacientes adultos com diversos tipos de câncer. Os pacientes com anemia foram sub-classificados como tendo anemia ferropriva (AF) (hipoferremia e hipoferritinemia), Anemia de Doença Crônica (ADC) (hipoferremia e ferritina sérica normal ou aumentada) e Anemia Relacionada ao Câncer (ARC) (sem anormalidades no metabolismo do ferro). Para a avaliação da eritropoiese foram realizados hemograma completo e dosagem de eritropoetina; para a avaliação do metabolismo do ferro: determinações do ferro sérico, capacidade de ligação do ferro à transferrina, ferritina e receptores solúveis da transferrina (sTfR) e Índice sTfR/ log ferritina; para a avaliação da resposta inflamatória: dosagens de Proteína C Reativa (PCR), Interleucinas 1? e 6, Fator de Necrose Tumoral alfa, neopterina e pro-hepcidina séricas e determinação da Velocidade de Hemossedimentação das hemáceas(VHS). Dentre os pacientes com câncer 34,8 % apresentaram anemia. O tipo de anemia mais freqüente foi a ARC (37%) seguido pela ADC (20,7%) e finalmente pela AF (17%). Os níveis de PCR, IL-6, VHS e IL-1? foram significativamente superiores em pacientes com ADC quando comparados com AF, ARC ou em pacientes sem anemia. Os níveis de Pró-Hepcidina, TNF-? e neopterina não foram diferentes entre os grupos de pacientes com câncer, mas foram superiores quando comparados a um grupo de indivíduos normais. O efeito negativo de fatores inflamatórios sobre a eritropoiese foi observado no grupo de pacientes com ADC, onde houve correlação inversa entre hemoglobina e ferritina sérica, PCR e IL-6. Não houve correlação entre os níveis de pro-hepcidina com o grau de anemia ou com parâmetros relacionados ao metabolismo de ferro. De acordo com nossos resultados, embora não tenha sido possível demonstrar uma relação direta entre inflamação e alterações no metabolismo do ferro, a atividade inflamatória presente em todos os grupos de pacientes, aparentemente está especialmente relacionada à patogênese da ADC. A hepcidina é reconhecida como importante reguladora e como principal mediadora das alterações do metabolismo do ferro na ADC. No presente trabalho, usando um ensaio imunoenzimático comercial, não foi possível demonstrar o significado fisiológico da pro-hepcidina na anemia em pacientes com câncer / Abstract: Patients with cancer present several hematological alterations, and anemia is the most common of them. Anemia ethiology is multifactorial and can be related or not to the disease (bleeding, hemolysis, bone marrow infiltration), to the type and stage of the tumor and also to the therapy. Hemoglobin reduction can be consequent to alteration in production and response of erythroid precursor to erythopoietin, releasing of cytokines and nutritional deficiency. The objective of this study was to evaluate some aspects related to inflammatory activity on iron metabolism and erythropoiesis in patients with solid tumors. Blood samples from 402 patients were analyzed and those patients with anemia were subdivided into: iron deficient anemia (IDA) (hypoferremia and hypoferretinemia), chronic disease anemia (CDA) (hypoferremia and serum ferritin normal or elevated) and anemia related to cancer (ARC) (no iron abnormalities). Erythropoiesis was assessed by complete blood cell count and erythropoietin determinations; iron metabolism by serum iron, total iron binding capacity, serum ferritin, soluble transferrin receptors (sTfR) and sTfR/log ferritin indices; inflammatory response by C-Reactive protein (CRP), interleukins 1β e 6, tumor necrosis factor α (TNF- α), neopterin, pro-hepcidin and erythrocyte sedimentation rate (ESR). Anemia was observed in 34.8% of cancer patients. The majority of patients presented ARC (37%), followed by CDA (20.7%) and IDA (17%). CRP, IL-6, ESR and IL-1β levels were significantly higher in patients with CDA than in IDA, ARC and cancer without anemia. Pro-hepcidin, TNF-α and neopterin values were not different among anemic patients groups, although higher levels of neopterin had been observed in patients with cancer when compared to normal individuals. The negative effect of inflammatory factors on erythropoiesis represented by inverse correlations between hemoglobin and serum ferritin, CRP and IL-6 were observed in ADC group. There was correlation neither between pro-hepcidin levels and anemia degree nor between pro-hepcidin and iron metabolism parameters. According to our results, although it was not possible to demonstrate a direct correlation between inflammation and iron metabolism alterations, the inflammatory activity apparently is related to ADC pathogenesis. Hepcidin is an important regulator of iron metabolism and has been recognized as the main mediator of iron metabolism alterations in CDA. In our study, by using ELISA test, it was not possible to show the participation of pro-hepcidin in anemia in cancer patients / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Orientador: Heitor Moreno Junior / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T03:27:07Z (GMT). No. of bitstreams: 1 Barbaro_NataliaRuggeri_M.pdf: 2773445 bytes, checksum: e0d750017009a49d1fe96a9e75554e89 (MD5) Previous issue date: 2013 / Resumo: A inflamação tem sido associada à hipertensão arterial e lesões de órgãos-alvo, sendo que elevados níveis de biomarcadores inflamatórios como a interleucina 6 (IL-6), 10 (IL-10), 1? (IL-1?), fator de necrose tumoral alfa (TNF-?) e a proteína C reativa ultra sensível (PCR-US) têm sido relacionados à doenças cardiovasculares. Estes biomarcadores inflamatórios estão envolvidos na rigidez arterial, um importante fator de risco cardiovascular. De fato, a hipertensão arterial resistente (HAR) possui um prognóstico desfavorável atribuído ao não controle pressórico e a outros fatores de risco associados. Entretanto, ainda não foi estabelecido o perfil dessas citocinas na HAR, também não se conhece o potencial impacto desses biomarcadores inflamatórios na rigidez arterial. Este estudo transversal em 32 pacientes com HAR, 20 com hipertensão de grau leve a moderado (HAS) e 20 normotensos (NT) teve como objetivo avaliar a relação de marcadores inflamatórios com a rigidez arterial em pacientes hipertensos resistentes. Foram avaliadas as medidas de pressão arterial de consultório e rigidez arterial, através da velocidade de onda de pulso (VOP). As concentrações plasmáticas de IL-10, IL-6, IL-1? e TNF-? foram determinadas por ELISA e PCR-US por nefelometria. O teste de Kruskal-Wallis foi utilizado para comparar os níveis de IL-6, TNF- ? e PCR-US, assim como os valores de VOP, enquanto o teste Qui-quadrado foi utilizado para avaliação das variáveis IL-10 (<1,0 and >1,0pg/mL) e IL-1? (<0,012 and >0,012 pg/mL). Análise de regressão linear múltipla foi conduzida para testar se havia associação independente dos marcadores inflamatórios (IL-6, TNF-?, IL-1? e PCR-US) com a VOP. Os grupos não apresentaram diferenças de idade, gênero e IMC. A VOP foi maior nos pacientes HAR (10.5 ± 2.2m/s), seguidos por pacientes HAS (8.7±2.1m/s) e indivíduos NT (7.2± 1.0m/s) (p<0.05). Os níveis de TNF-? (média; (95% CI)) foram significativamente maiores nos grupos HAR: 3,3 (2,19 -4,42pg/mL) e HAS: 3,09 (2,61-3,56) comparados aos NT 1,94 (1,48 - 2,39) (p<0.05), mas não houve diferença entre níveis de IL-6. O grupo de HAR teve maior proporção de sujeitos com níveis elevados de IL-10 (78.1% de IL-10 >1.0 pg/mL) comparados aos HAS (50%) e NT (20%). Além disso, a proporção de pacientes HAR com IL-1? aumentada também foi maior (100%) comparada aos HAS (45%) e NT (25%). Finalmente, apenas a IL-1? foi independentemente associada a VOP (p<0,001; R2= 0,49; ?=0,079). Pacientes HAR apresentaram níveis aumentados de citocinas inflamatórias (TNF-?, IL-1? and IL-10), assim como rigidez arterial. Em adição, a concentração de IL-1? é um fator de risco independente para rigidez arterial / Abstract: Inflammation has been associated with hypertension and target organ damage and increased levels of inflammatory biomarkers such as interleukin-6 (IL-6), 10 (IL-10), 1? (IL-1?), tumor necrosis factor-? (TNF-?) and high sensitive C protein (hs-CRP) have been described in cardiovascular diseases. These inflammatory biomarkers are implicated in arterial stiffness, an important cardiovascular risk factor. Indeed, resistant hypertension (RHTN) leads to unfavorable prognosis attributed to poor blood pressure (BP) control and other cardiovascular risk factors. However, the potential impact of these inflammatory biomarkers on arterial stiffness in RHTN subjects has not been demonstrated. A cross-sectional study was performed in 32 RHTN, 20 mild to moderate hypertensive patients (HTN) and 20 normotensive (NT) subjects. Office BP and arterial stiffness, assessed by pulse wave velocity (PWV) were evaluated. Indeed, IL-10, IL-6, IL-1? and TNF-? were determined by ELISA and hs-CRP by nephelometry. Kruskal-Wallis was used to compare levels of IL-6, TNF- ? and hs-CRP as well as values of PWV, whereas Chi-square was used to evaluated IL-10 (<1.0 and >1.0pg/mL) and IL-1? (<0.012 and >0.012 pg/mL). Multiple linear regression analysis tested the association of inflammatory biomarkers (IL-10, TNF-?, IL-1? and hs-CRP) with PWV. No differences were observed between the 3 groups with respect to age, gender and BMI. PWV was increased in RHTN (10.5 ± 2.2m/s) followed by HTN patients (8.7±2.1m/s) compared to NT individuals (7.2± 1.0m/s) (p<0.05). TNF-? levels (mean; (95% CI)) were significantly higher in RHTN 3.3 (2.19 - 4.42pg/mL) and HTN: 3.09 (2.61-3.56) than NT subjects 1.94 (1.48 - 2.39) (p<0.05) but no differences were observed regarding IL-6 levels. RHTN group had higher frequency of subjects with increased levels of IL-10 (78.1% of IL-10 >1.0 pg/mL) compared with HTN (50%) and NT (20%). Moreover, RHTN had higher frequency of subjects with increased levels of IL-1?(100%) compared to HTN (45% ) and NT (25%). Finally, IL-1? was independently associated to PWV (p<0.001; R2= 0.49; ?=0.079). Taken together, RHTN subjects presented increased levels of inflammatory cytokines (TNF-?, IL-1? and IL-10) as well as of arterial stiffness. In addition, we found that IL-1? levels are an independent risk factor for arterial stiffness / Mestrado / Farmacologia / Mestra em Farmacologia

CD23, the low affinity IgE receptor, is expressed by various cell types and has numerous functions depending on the form of the protein, its interaction with various ligands and the type of cell involved. CD23 is pivotal in the regulation of IgE, with the soluble form involved in up-regulation, while the membrane bound form is involved in the down-regulation. It is clear why it is believed to be a central molecule in allergic responses, and a therapeutic target for the treatment of allergic disease. In this study a recombinant form of the entire extracellular domain of the protein, exCD23, was produced by PCR cloning and expressed in E. coli. His•Tag™s were introduced onto the C-terminus and N-terminus, respectively, in order to simplify the purification procedure. After renaturation and purification, the recombinant exCD23 bound IgE, indicating its activity. From the IgE binding studies it was established that the position of the tag did not influence the binding. GST•Tagged™ exCD23 was also produced in an attempt to increase the solubility of the recombinant protein, but this proved unsuccessful. Butyrate differentiated EoL-1 cells were treated with the Nterminal His•Tagged™ exCD23, and the protein appeared to suppress the secretion of the constitutively expressed cytokines, especially IL-8 and IFN- , when compared to untreated cells. In addition, treatment of the EoL-1 cells with exCD23 had a significant proliferative effect, but could not induce differentiation of this cell line into mature eosinophilic-like cells.

Thesis (D.Sc.D.)--Boston University, Henry M. Goldman School of Dental Medicine, 1999. / Includes bibliographical references (leaves 90-106). / Tooth eruption provides an excellent model to examine osseous metabolism as bone resorption (occlusal area), and formation (apical area), occur simultaneously and are spatially separated. Monocytes are thought to play an important role in regulation of bone metabolism. The goal of this study was to examine recruitment of monocytes to bone undergoing developmental remodeling in C57BL/6J mice. To account for potential mechanism for monocyte recruitment, we investigated expression of monocyte chemoattractant protein-1 (MCP-1) in tissues surrounding an erupting tooth. TNF and IL1 are potent stimulators of bone resorption. Recent evidence shows that these proinflammatory cytokines are expressed during embryogenesis and may participate in developmental tissue remodeling. To establish their role in tooth eruption and bone remodeling, we carried out experiments using mice with genetic deletions of TNFR1/IL-1R1 or TNFR1/TNFR2. Mandibles were obtained from animals sacrificed at various time points from birth to 14 days of age. Histological sections were stained using immuno/histochemistry to identify mononuclear phagocytes, osteoclasts, MCP-1-positive and apoptotic cells. The results demonstrated that a significant time-dependent increase in recruitment of monocytes in the occlusal area (bone resorption) at days 5 and 9 was associated with significant increase in number of osteoclasts at similar time points. In contrast, in the apical area (bone formation), a significant time-dependent increase in monocyte recruitment was coupled with a decrease in number of osteoclasts, found in high numbers at earliest time points (up to day 3 postnatally). The number of MCP-1 positive cells also increased with time in both areas and was generally proportional to the recruitment of mononuclear phagocytes. Osteoblasts were the principal bone cell type expressing MCP-1. Our results suggest that monocytes have different functional roles in areas of bone resorption and bone formation. Furthermore, the expression of MCP-1 is developmentally regulated and may provide mechanistic basis to explain the recruitment of monocytes. Functional deletion of TNFR1/IL-1R1 resulted in later onset of molar eruption. However, histological findings showed that only monocyte physiology in the occlusal connective tissue was affected by loss of TNFR1/IL-1R1 signaling. Increased number of monocytes in the area was observed during tooth eruption through subgingival connective tissue (day 13). Presence of monocytes in the dental follicle or presence of osteoclasts at the adjacent bone surface was not altered. Deletion of TNFR1/R2 affected recruitment/function/survival of monocytes and rate of apoptosis only in the apical area during intraosseous stage of tooth eruption (day 9). No changes in monocyte or osteoclast markers were noted in the occlusal area. Loss of TNFR1/R2 signaling had no effect on the rate of molar eruption. Lack of striking differences between the experimental and the wild type groups indicates that TNF and IL-1 do not play a critical role during tooth eruption and remodeling of surrounding bone, supporting the suggested hypothesis that tooth eruption is a redundantly regulated process.

Atherogenesis involves the activation of endothelial cells and the egress of atherogenic T lymphocytes and monocytes into the intima. Exercise training contributes to the arrest and even reversal of atherosclerosis by modifying risk factors and by inducing an atheroprotective phenotype in endothelial cells and T cells.