The aim of the study was to investigate the association between depression and low bone mineral density (BMD) in premenopausal females. The rationale for the study was that depression is often characterized by cortisol hypersecretion. The role of cortisol includes effects on bone metabolism and the immune system: cortisol is a bone resorption agonist through its support of osteoclastogenesis. The release of pro-inflammatory cytokines, (especially IL-1, IL-6 and TNF-alpha) which induce cortisol secretion, also pushes the balance of bone remodelling in favour of resorption, consequently causing loss of bone mineral density. Significant results have been reported in studies of various groups across the USA, Europe and Asia, indicating a causal role for depression in osteoporosis. However, some studies could not support this association. With both osteoporosis and depression representing growing public health concerns in South Africa, the aim of this study was to examine the association between depression and loss of BMD in a South African sample with varying levels of depression. The study was approached from two starting points: the first used low BMD as the departure point and the second was undertaken from the diagnosis of depression. This was achieved by first investigating women where the primary concern was possible low BMD (referred to as Study 1) and secondly by assessing women whose primary diagnosis was clinically confirmed major depression (Study 2). Study 1 involved investigation of BMD in a volunteer-based sample of 40 premenopausal women drawn from three different sources. All volunteers underwent a DEXA scan, were assessed for depression and supplied saliva for cortisol analysis. Study 2 examined the BMD of five psychiatric patients diagnosed with severe, recurrent major depression and four healthy controls. These volunteers were required to undergo the same testing as subjects in Study 1. In addition, blood and urine samples were taken to examine bone turnover markers (bone specific alkaline phosphate, osteocalcin, urine pyridinoline cross-linked C-telopeptide and deoxypyridinoline). The pro-inflammatory status of the psychiatric patients was compared to reference ranges. The latter served as a small exploratory study and an introduction to further avenues of research. Study 1 revealed no clear general association between depression and bone density on DEXA scores. However, a correlation was found between left femoral neck BMD and depression in those women with low BMD only. Significant differences were found though between subjects with normal and low BMD in terms of body mass index (BMI) and contraception use. Study 2 on the other hand, indicated a trend of association between depression and low BMD: subjects suffering with severe major depression were noted to have lower bone density (on DEXA) and higher bone turnover (as measured by markers of bone turnover) as well as higher cortisol levels than healthy controls. In addition, depressed subjects exhibited elevated IL-1-alpha levels but normal TNF-alpha levels when compared to normative data. In conclusion, the study indicated that the effect of depression on bone density is dependent on the intensity and duration of depression. IL-1-alpha and cortisol may be instrumental in this loss of BMD. / Dissertation (MSc)--University of Pretoria, 2008. / Physiology / unrestricted

O Diabete tipo 2 é uma síndrome heterogênea caracterizada por resistência à insulina e /ou diminuição relativa da função secretora das células &#946; pancreáticas. Os Diabéticos têm risco maior de desenvolver aterosclerose, que é uma doença inflamatória crônica que envolve a resposta imune. Os TLRs sinalizadores da resposta imune inata que ativam vias que participam na regulação da inflamação podem estar associados com a patogênese da aterosclerose. Além disso, são capazes de induzir a resistência à insulina. Estudos sugerem que a inflamação é um fator chave na aterogênese em diabéticos tipo 2. Citocinas pró-inflamatórias, como a IL-6 e o TNF-&#945;, são produzidas pelo tecido adiposo em grande quantidade em indivíduos obesos, especialmente em pacientes com DM2. Este estudo tem por objetivo avaliar a associação dos genes do TLR2, TLR4, TNF-&#945; e IL-6 com o diabetes tipo 2 e com a aterosclerose. Foram selecionados sessenta e um indivíduos DM2 e trinta e nove indivíduos normoglicêrmicos (grupo controle) na seção de Coronárias do Instituto Dante Pazzanese de Cardiologia (São Paulo, Brasil). O ultra-som de carótida foi utilizado para avaliar a presença de aterosclerose. Os polimorfismos dos genes TLR4 (Thr399lle), TLR2 (Arg753Gln), and IL6 (-174G>C) foram identificados pela PCR-RFLP. O polimorfismo dos genes TLR4 (Asp299Gly) and TNF-&#945; (-308G>A) foram detectados por HRM. A expressão do mRNA nos leucócitos do sangue periférico foi mensurado pela PCR em tempo real utilizando o gene GAPD como gene referência. No grupo diabéticos, indivíduos portadores do alelo IL-6 -174C apresentaram colesterol total, VLDL-C e triglicérides que os portadores do genótipo GG (respectivamente, p=0,007, p=0,006 e p= 0,030). A expressão de TLR4 foi maior em indivíduos do grupo diabéticos que no grupo controle (p=0,029). Em DM2, o genótipo TNF-&#945; -308GG foi associado com o aumento da expressão do mRNA do gene TNF-&#945; (p=0,031) e maiores concentrações de fibrinogênio que os portadores do alelo -308A (genótipo GA+AA) (p=0,020). Em indivíduos com aterosclerose o alelo TLR4 299Gly foi associado com altas concentrações de glicemia pós 75g de glicose (p=0,012). E portadores do alelo IL6 -174C apresentaram maiores concentrações de colesterol total e LDL-C que nos portadores do genótipo GG (ambos p<0,001). O polimorfismo -174G>C (alelo C) e o DM2 mostraram serem fatores de risco para a aterosclerose (odds ratio respectivamente: 3,0 and 16,962). E, o sexo masculino e a menopausa mostraram serem fatores de risco para o DM2. / Type 2 diabetes is an heterogeneous syndrome characterized by the resistance to insulin and/or relative decrease of the secretion of insulin of the pancreatic &#946; cells. T2DM are in high risk of develop atherosclerosis, that is a chronic inflammatory disease involving immune response. Many existing in the atherosclerotics plaques the TLRs are signaling for immune innate response, that activate inflammatory cells involved in progression of atherosclerotic disease. TLRs can be also associated with induction of the resistance to insulin. Many studies have been suggesting that inflammation is a key factor of the progression of atherosclerotic disease in type 2 diabetes. Proinflammatory cytokines, such as IL-6 and TNF-&#945;, are produced by the adipose tissue in high concentration in obese individuals, mainly among patients with T2DM. This study aims to investigate the relationship between TLR2, TLR4, TNF-&#945; and IL-6 gene expression and polymorphisms with T2DM and atherosclerosis. Sixty-one T2DM and thirty nine normoglycemic (control group) individuals were selected at the Coronary Session of the Instituto Dante Pazzanese de Cardiologia (Sao Paulo, Brazil). Carotid artery ultrassonography was used to evaluate the atherosclerotic status. Polymorphisms of TLR4 (Thr399lle), TLR2 (Arg753Gln), and IL6 (-174G>C) genes were detected by PCR-RFLP. TLR4 (Asp299Gly) and TNFA (-308G>A) gene polymorphisms were detected by HRM. Blood leukocytes mRNA expression was measured by real time PCR using GAPD as a reference gene. In T2DM, individuals carrying IL6 -174C allele had higher total cholesterol, VLDL-c and triglycerides than the genotype GG carriers (respectively, p=0,007, p=0,006 and p= 0,030). TLR4 mRNA expression was higher in T2DM than in control individuals (p=0,029). In T2DM group, TNF-&#945; -308GG genotype was associated with increased TNF-&#945; mRNA expression levels (p=0,031) and higher fibrinogen levels than those carrying -308A allele (GA+AA genotypes) (p=0,020). In individuals with atherosclerosis TLR4 299Gly allele was associated with high post-test glucose levels (p=0,012). And carrying IL6 -174C allele had higher total cholesterol and LDL-C than the genotype GG carriers (both p<0,001). The polymorphism -174G>C and the T2DM was related with risk for atherosclerosis (odds ratio respectively: 3,0 and 16,962) and, the male sex and menopause was related with risk for T2DM (odds ratio respectively: 3,401 and 3,025).

Recent evidence from cancer research indicates that lactate exerts a suppressive effect on innate immune responses in cancer. This study investigated the mechanisms by which lactate suppresses macrophage pro-inflammatory responses. Macrophages [Raw 264.7 and bone marrow derived macrophages (BMDMs)] were treated with LPS in the presence or absence of lactate. Pro-inflammatory cytokines, NF-κB and YAP activation and nuclear translocation were examined. Our results show that lactate significantly attenuates LPS stimulated macrophage TNF-α and IL-6 production. Lactate also suppresses LPS stimulated macrophage NF-κB and YAP activation and nuclear translocation in macrophages. Interestingly, YAP activation and nuclear translocation are required for LPS stimulated macrophage NF-κB activation and TNFα production. Importantly, lactate suppressed YAP activation and nuclear translocation is mediated by GPR81 dependent AMKP and LATS activation which phosphorylates YAP, resulting in YAP inactivation. Finally, we demonstrated that LPS stimulation induces an interaction between YAP and NF-κB subunit p65, while lactate decreases the interaction of YAP and NF-κB, thus suppressing LPS induced pro-inflammatory cytokine production. Our study demonstrates that lactate exerts a previously unknown role in the suppression of macrophage pro-inflammatory cytokine production via GPR81 mediated YAP inactivation, resulting in disruption of YAP and NF-κB interaction and nuclear translocation in macrophages.

Background: Knee osteoarthritis (OA) is the most common form of arthritis in older adults. Knee OA is associated with limitations in physical function. Functional limitations are also associated with another geriatric condition, frailty. Frailty is characterized by reduced strength, endurance and physiological function. Purpose: The primary purpose of this study is to determine if there is a difference in radiographic or symptomatic knee OA severity between non-frail and pre-frail women with knee OA. Secondary objectives include: a) the relationship between radiographic and symptomatic OA severity with serum inflammatory cytokines, and b) if there is a difference in inflammatory cytokines between non-frail and pre-frail women with knee OA. Methods: We included 21 community-dwelling women with knee OA. Frailty was assessed using the Fried Frailty Phenotype. Knee OA severity was characterized by the Kellgren and Lawrence (KL) score and the Knee Injury and Osteoarthritis Outcome Questionnaire (KOOS). Inflammatory cytokines included serum interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis alpha (TNF α) and C reactive protein (CRP). Results: Data from 20 participants (66.1 [9.6] years, BMI 29.7 [4.9] kg/m2, non-frail=55%; pre-frail=45%) were analyzed. Radiographic severity was not different between frailty groups (p= 0.11). There was no difference in symptomatic knee OA severity, measured using the KOOS subscales, between frailty groups (p>0.17). Radiographic OA severity and inflammatory markers were not associated (p>0.30). There was a negative relationship between TNF α and self-reported pain (r=0.26), no relationships between inflammatory cytokines with any other KOOS sub-scales. Lastly, there was no difference in any inflammatory cytokines between non-frail and pre-frail groups. Conclusion: Despite the relatively young age, nearly 50% of our participants were pre-frail. Pre-frailty was unrelated to the severity of the knee OA, or inflammatory cytokines. TNF α may be involved in the experience of pain in these women. While it appears women with knee OA frequently demonstrate pre-frail status, more work is necessary to examine the link between these diseases. / Thesis / Master of Science (MSc) / Arthritis is a chronic disease that has a debilitating effect on the lives of more than 4.6 million Canadians. In 2015, the cumulative economic burden of osteoarthritis was 195.2 billion dollars and is expected to increase significantly in the next two years. Knee osteoarthritis is the most common form of arthritis in older adults. Knee osteoarthritis is associated with increased pain, decreased physical function and decreased quality of life (QOL). In vulnerable older adults increased exhaustion, decreased physical function and muscle loss can increase the risk of developing frailty. Frail older adults are at higher risk of adverse health outcomes such as falls, hospitalization and death. Previous research has shown that older adults with knee OA are at higher risk of developing frailty however, it is not understood what underlying mechanisms increase this risk. This thesis provides fundamental information aimed at understanding potential mechanisms associated with knee osteoarthritis and frailty in women. Our study found that despite their relatively young age, nearly half of the women with knee OA are pre-frail. This data shows that inflammatory cytokines in particular, tumor necrosis factor alpha is related to symptomatic knee osteoarthritis severity in particular, self-reported pain. Overall, early detection of frailty is important when managing this condition. These data suggest that chronic knee pain associated with OA may be a useful trigger for early assessments of frailty in women.

No description available.

No description available.

In the United States, over one third of adult women are obese, and one in eight women will be diagnosed with breast cancer in their lifetime. Obesity has been shown to be a risk factor for postmenopausal breast cancer and is associated with increased aggressiveness and poor prognosis regardless of menopausal status. However, the mechanisms involved in the relationship between obesity and breast cancer are still not fully understood. Wnt signaling is often elevated in breast tumors (~60%) and is suspected to play a key role in cancer development. It has been shown that inflammatory cytokines, such as TNF-α, IL-1β, IFNγ, are potential mediators in the regulation of Wnt-signaling. We hypothesize that the low-grade inflammatory state associated with obesity is present in human mammary tissue, stimulates Wnt activity, and thereby leads to the development of breast cancer. In this project, we propose to 1) characterize the inflammatory cytokine profile, including IFN-γ, IL-1β, IL-2, IL-6, IL-8, and TNF-α, in the mammary tissue of normal weight, overweight, and obese postmenopausal women using a high performance electrochemiluminescence immunoassay; 2) determine the influences of the obesity-induced pro-inflammatory cytokines on Wnt-signaling by examining gene expression of seven Wnt-signaling target genes using real-time PCR; and 3) define the causality between TNF-α, one of the mot critical inflammatory cytokines, and Wnt signaling by measuring the gene expression of the Wnt targets in samples from normal to overweight and obese postmenopausal women treated with anti-TNF-α antibody or TNF-α recombinant protein respectively. We expect to define a novel mechanism that obesity mediates the development of postmenopausal breast via inflammation-driven Wnt signaling.

African monkey species are natural hosts of simian hemorrhagic fever virus (SHFV) and develop persistent, asymptomatic infections. SHFV was previously shown to also cause a rapid onset fatal hemorrhagic fever disease in macaques. Infection of macaques with a new isolate of SHFV from persistently infected baboon sera, that showed high nucleotide identity with the lab strain LVR, resulted in viremia, pro-inflammatory cytokine and tissue factor production, and symptoms of coagulation defects. Primary macrophages and myeloid dendritic cell cultures from disease-susceptible macaques efficiently replicated SHFV and produced pro-inflammatory cytokines, including IL-6 and TNF-α, as well as tissue factor. Cells from disease resistant baboons produced low virus yields and the immunomodulatory cytokine IL-10. IL-10 treatment of macaque cells decreased IL-6 levels but had no effect on TNF-α levels, tissue factor or virus production suggesting that IL-10 plays a role in modulating immunopathology in disease-resistant baboons but not in regulating the efficiency of virus replication. SHFV is a member of the family Arteriviridae. The SHFV genome encodes 8 minor structural proteins. Other arteriviruses encode 4 minor structural proteins. Amino acid sequence comparisons suggest that the four additional SHFV minor structural proteins resulted from gene duplication. A full-length infectious clone of SHFV was constructed and produced virus with replication kinetics comparable to the parental virus. Mutant infectious clones, each with the start codon of one of the minor structural proteins substituted, were analyzed. All eight SHFV proteins were required for infectious virus production. The SHFV nonstructural polyprotein is processed into the mature replicase proteins by several viral proteases including papain-like cysteine proteases (PLPs). Only one or two PLP domains are present in other arteriviruses but SHFV has three PLP domains. Analysis of in vitro proteolytic processing of C- and N-terminally tagged polyproteins indicated that the PLP in each of the three SHFV nsp1 proteins is active. However, the nsp1α protease is more similar to a cysteine protease than a PLP. Analysis of the subcellular localization of the three SHFV nsp1 proteins indicated they have divergent functions.

O carcinoma epidermoide de cabeça e pescoço (HNSCC) é uma das neoplasias mais frequentes e geralmente está associado a inflamação crônica e níveis sistêmicos de citoquinas. Seus principais fatores de risco são a exposição ao tabaco e álcool. Publicações recentes sugerem que a infecção por papiloma vírus HPV está relacionada com a tumorigênese de cabeça e pescoço e pode alterar o perfil e o desfecho deste tumor. A transição epitelial-mesenquimática (EMT) é um processo importante durante a tumorigênese pelo qual células epiteliais obtêm um fenótipo migratório e invasivo. Os efeitos da infecção por HPV e de citocinas inflamatórias neste processo ainda não são bem compreendidos em HNSCC. O presente estudo teve como objetivo transfectar células normais e neoplásicas com os genes E6/E7 de HPV16 e investigar os mecanismos pelos quais a EMT é ativada por citocinas inflamatórias e por infecção por HPV. Taxas de proliferação, viabilidade, migração e invasão celular induzidas por IL-6, TNF-a e TGF-beta foram avaliadas em linhagens celulares derivadas de queratinócitos normais e de carcinoma de língua (HaCat e SCC25, respectivamente) transfectadas com os genes E6/E7 de HPV16 e a expressão dos marcadores relacionados a EMT foram analisados por PCR em tempo real nas linhagens SCC25, HaCat e FaDu (carcinoma de faringe). Os resultados sugerem que o HPV modificou a morfologia das linhagens normal e tumoral. Na linhagem HaCat, o ambiente inflamatório estimulou modificações importantes para o desenvolvimento de condições patológicas. A inserção dos genes E6/E7 de HPV16 diminuiu a proliferação e a viabilidade na linhagem HaCat e o ambiente inflamatório não modificou a resposta iniciada pelo HPV. Na linhagem SCC25 a inserção de HPV em associação com inflamação reduziu a progressão da EMT, ao contrário do que foi observado na linhagem FaDu. A relação de HPV com inflamação levando a progressão de EMT é controversa e dependente do sítio anatômico em tumores de cabeça e pescoço / Head and neck squamous cell carcinoma (HNSCC) is one of the most frequent neoplasias and is often associated with chronic inflammation and systemic cytokine levels. Its main risk factors are tobacco and alcohol exposition. Recent publications suggest that the infection by human papillomavirus (HPV), especially high-risk types, is related to head and neck tumorigenesis and may alter the tumor profile and outcome. Epithelial-mesenchymal transition (EMT) is an important process during tumorigenesis by which epithelial cells gain a migratory and invasive phenotype. The effects of HPV infection and inflammatory cytokines on this process are still not well understood in HNSCC. The present study aimed to transfect normal and neoplastic cells with E6/E7 genes of HPV type 16, and to investigate the mechanisms by which EMT is activated by inflammatory cytokines and by HPV infection. Proliferation, viability, migration and cell invasion rates induced by IL-6, TNF-a and TGF-beta cytokines will be also evaluated in cell lines derived from normal keratinocyte and tongue carcinoma (HaCat and SCC25, respectively) transfected with HPV16 E6/E7 genes and the expression of the EMT-related markers were analyzed by real-time PCR in the SCC25, HaCat and FaDu (pharyngeal carcinoma). The results suggest that HPV modified the morphology of normal and tumor cell lines. In the HaCat cell line, the inflammatory environment stimulated important modifications for the development of pathological conditions. Insertion of HPV16 E6/E7 genes decreased proliferation and viability in the HaCat and the inflammatory environment did not modify the HPV-initiated response. In the SCC25 cell line the insertion of HPV in association with inflammation reduced the progression of EMT, unlike was observed in FaDu. The relationship of HPV with inflammation leading to progression of EMT is controversial and depends on anatomical site in head and neck carcinoma

A hanseníase é uma doença crônica causada por Mycobacterium leprae e apresenta diversas formas clínicas. O entendimento da interação parasita-hospedeiro na hanseníase evidenciou que ocorre a persistência assintomática do patógeno, caracterizando um estado de latência. Os fatores mais importantes relacionados com a permanência do patógeno são: a patogenicidade do agente infeccioso e o perfil da resposta imune, no qual os eventos de migração celular, produção de citocinas, as células efetoras e reguladoras são extremamente relevantes. As células T reguladoras (Treg) desempenham papel central na regulação da resposta imune em infecções crônicas o que favorece a persistência do patógeno. A importância de células T reguladores na hanseníase ainda é pouco conhecida. Neste trabalho investigou-se a presença de células T reguladoras em lesões e sangue periférico de indivíduos com hanseníase. Inicialmente avaliou-se a proliferação e a produção de citocinas por células mononucleares do sangue periférico (PBMC) de pacientes com hanseníase. Os resultados evidenciaram que não há diferenças quanto à proliferação de células T e produção de IFN-&#947; e TNF-&#945; por células desses pacientes, mas a produção de IL-4 e IL-5 foi detectada apenas entre os pacientes com hanseníase virchoviana. Em relação à presença de células T reguladoras, os resultados evidenciaram aumento no número de linfócitos T CD4+CD25+FoxP3+ no sangue periférico de pacientes com hanseníase virchoviana. As células T reguladoras dos pacientes com hanseníase apresentaram elevada expressão de moléculas co-inibitórias PD-1, CTLA-4, GITR e ICOS. De modo relevante, as células T CD4+CD25+ isolados de pacientes com hanseníase virchoviana apresentaram maior atividade supressora quando comparado às células isoladas de pacientes com hanseníase tuberculóide. As células T CD4+CD25+ de pacientes com hanseníase virchoviana inibiram a proliferação de PBMC alogênico e a produção de IFN-&#947; e TNF-&#945;. Os resultados demonstraram também que nas amostras de lesão de pele de pacientes com hanseníase virchoviana há acúmulo de células CD25+ produtoras de IL-10 e TGF-&#946;, enquanto que estas células não foram detectadas nas lesões de pacientes com hanseníase tuberculóide. Dessa forma, os resultados descritos indicam que pacientes com hanseníase virchoviana apresentam aumento no número de células T reguladoras circulantes e no infiltrado inflamatório, e estas células apresentaram maior atividade supressora. O acúmulo de células T reguladoras no sítio da infecção pode ser correlacionado com o controle da resposta imune e conseqüente persistência de M. leprae. / Leprosy is caused by Mycobacterium leprae and its clinical features depend on the host immune background. The understanding of parasite-host interactions in leprosy have highlighted asymptomatic persistence of the pathogen, which indicates that this infection becomes latent. The most important factors related to the permanence of pathogens are: the pathogenicity of the infectious agents; the profile of the immune response developed by the host whose events of cellular migration, cytokines production, and the effector and regulatory cells are extremely relevant. The regulatory T cells (Treg) seem to play a central role in the regulation of the immune response in chronic infections, which favors the persistence of the pathogen. Herein, we analyzed the relation between tuberculoid and lepromatous leprosy with the presence and function of T regulatory cells from peripheral blood mononuclear cells (PBMC) and skin lesions from these patients. First, the proliferation and cytokine production of PBMC isolated from leprosy patients were analyzed. We did not observe any difference in the proliferation ability or IFN-&#947; and TNF-&#945; release; however, the production of IL-4 and IL-5 was detected only in patients with lepromatous leprosy. Furthermore, T CD4+CD25+FoxP3+ cells were detected in the PBMC of patients with leprosy and these cells from lepromatous patients showed high expression of co-inhibitory molecules such as PD-1, GITR, CTLA-4 and ICOS. T CD4+CD25+cells isolated from patients with lepromatous leprosy were significantly more suppressive than the cells obtained from tuberculoid patients. In addition, TCD4+CD25+ cells isolated from patients with lepromatous leprosy inhibited allogeneic PBMC proliferation and their production of IFN-&#947; and TNF-&#945;. The results also demonstrated that IL- 10 and TGF-ß were co-expressed with CD25+ cells at the inflammatory infiltrate of skin lesions from lepromatous patients, but similar results were not detected among tuberculoid patients. Thus, these results indicate that lepromatous leprosy patients have an enhanced presence of Treg cells with a suppressive ability in the blood and in the inflammatory infiltrate. The accumulation of Treg cells at the infection sites might be associated to the control of immune response and consequently to Mycobacterium leprae presistence.