The pathogenesis of oral lichen planus

Both antigen-specific and non-specific mechanisms may be involved in the pathogenesis of oral lichen planus (OLP). Antigen-specific mechanisms in OLP include antigen presentation by basal keratinocytes and antigen-specific keratinocyte killing by CD8(+) cytotoxic T-cells. Non-specific mechanisms include mast cell degranulation and matrix metalloproteinase (MMP) activation in OLP lesions. These mechanisms may combine to cause T-cell accumulation in the superficial lamina propria, basement membrane disruption, intra-epithelial T-cell migration, and keratinocyte apoptosis in OLP. OLP chronicity may be due, in part, to deficient antigen-specific TGF-beta1-mediated immunosuppression. The normal oral mucosa may be an immune privileged site (similar to the eye, testis, and placenta), and breakdown of immune privilege could result in OLP and possibly other autoimmune oral mucosal diseases. Recent findings in mucocutaneous graft-versus-host disease, a clinical and histological correlate of lichen planus, suggest the involvement of TNF-alpha, CD40, Fas, MMPs, and mast cell degranulation in disease pathogenesis. Potential roles for oral Langerhans cells and the regional lymphatics in OLP lesion formation and chronicity are discussed. Carcinogenesis in OLP may be regulated by the integrated signal from various tumor inhibitors (TGF-beta1, TNF-alpha, IFN-gamma, IL-12) and promoters (MIF, MMP-9). We present our recent data implicating antigen-specific and non-specific mechanisms in the pathogenesis of OLP and propose a unifying hypothesis suggesting that both may be involved in lesion development. The initial event in OLP lesion formation and the factors that determine OLP susceptibility are unknown.

Adipose tissue as an endocrine organ

Adipose tissue is a highly active endocrine organ secreting a range of soluble products with both local and distant actions. These hormones have important roles in metabolism, reproduction, cardiovascular function and immunity. It is now evident that adipose endocrine function directly influences other organ systems, including the brain, liver and skeletal muscle. The endocrine function of adipose tissue is significantly regulated by nutritional status, and both are inextricably linked to the energy storage role of adipose tissue. This chapter highlights the endocrinology of adipose tissue by concentrating on functional aspects of the secreted products. The data of particular relevance to humans are highlighted, and areas in need of future research are suggested.

TACE inhibition: a new approach to treating inflammation

Clinical trials showing the benefits of reducing the effects of TNF-alpha in rheumatoid arthritis have highlighted the key role of the cytokine TNF-alpha in this inflammatory condition. A new approach to reducing the effects of TNF-alpha is to decrease its synthesis by inhibiting TNF-alpha converting enzyme with GW3333. In rat models of arthritis, GW3333 has some beneficial effects. Further longer-term studies of GW3333 in animal models are required to determine whether its benefit is maintained. TACE inhibition may represent a new approach to treating inflammation.

Mast cells and oral inflammation

Mast cells are mobile granule-containing secretory cells that are distributed preferentially about the microvascular endothelium in oral mucosa and dental pulp. The enzyme profile of mast cells in oral tissues resembles that of skin, with most mast cells expressing the serine proteases tryptase and chymase. Mast cells in oral tissues contain the pro-inflammatory cytokine tumour necrosis factor-alpha in their granules, and release of this promotes leukocyte infiltration during evolving inflammation in several conditions, including lichen planus, gingivitis, pulpitis, and periapical inflammation, through induction of endothelial-leukocyte adhesion molecules. Mast cell synthesis and release of other mediators exerts potent immunoregulatory effects on other cell types, while several T-lymphocyte-derived cytokines influence mast cell migration and mediator release. Mast cell proteases may contribute to alterations in basement membranes in inflammation in the oral cavity, such as the disruptions that allow cytotoxic lymphocytes to enter the epithelium in oral lichen planus. A close relationship exists among mast cells, neural elements, and laminin, and this explains the preferential distribution of mast cells in tissues. Mast cells are responsive to neuropeptides and, through their interaction with neural elements, form a neural immune network with Langerhans cells in mucosal tissues. This facilitates mast cell degranulation in response to a range of immunological and non-immunological stimuli. Because mast cells play a pivotal role in inflammation, therapies that target mast cell functions could have value in the treatment of chronic inflammatory disorders in the oral cavity.

A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations

The conjugation of antigens with ligands of pattern recognition receptors (PRR) is emerging as a promising strategy for the modulation of specific immunity. Here, we describe a new Escherichia coli system for the cloning and expression of heterologous antigens in fusion with the OprI lipoprotein, a TLR ligand from the Pseudomonas aeruginosa outer membrane (OM). Analysis of the OprI expressed by this system reveals a triacylated lipid moiety mainly composed by palmitic acid residues. By offering a tight regulation of expression and allowing for antigen purification by metal affinity chromatography, the new system circumvents the major drawbacks of former versions. In addition, the anchoring of OprI to the OM of the host cell is further explored for the production of novel recombinant bacterial cell wall-derived formulations (OM fragments and OM vesicles) with distinct potential for PRR activation. As an example, the African swine fever virus ORF A104R was cloned and the recombinant antigen was obtained in the three formulations. Overall, our results validate a new system suitable for the production of immunogenic formulations that can be used for the development of experimental vaccines and for studies on the modulation of acquired immunity.

Risk factors for metabolic bone disease in Crohn's disease patients

Background: The aim was to evaluate the presence of metabolic bone disease (MBD) in patients with Crohn’s disease (CD) and to identify potential etiologic factors. Methods: The case–control study included 99 patients with CD and 56 controls with a similar age and gender distribution. Both groups had dual-energy x-ray absorptionmetry and a nutritional evaluation. Single nucleotide polymorphisms at the IL1, TNF-a, LTa, and IL-6 genes were analyzed in patients only. Statistical analysis was performed using SPSS software. Results: The prevalence of MBD was significantly higher in patients (P ¼ 0.006). CD patients with osteoporosis were older (P < 0.005), small bowel involvement and surgical resections were more frequent (P < 0.005), they more often exhibited a penetrating or stricturing phenotype (P < 0.05), duration of disease over 15 years (P < 0.005), and body mass index (BMI) under 18.5 kg/m2 (P < 0.01) were more often found. No association was found with steroid use. Patients with a Z-score < 2.0 more frequently had chronic active disease (P < 0.05). With regard to diet, low vitamin K intake was more frequent (P ¼ 0.03) and intake of total, monounsaturated, and polyunsaturated fat was higher in patients with Z-score < 2.0 (P < 0.05). With respect to genetics, carriage of the polymorphic allele for LTa252 A/G was associated with a higher risk of osteoporosis (P ¼ 0.02). Regression analysis showed that age over 40 years, chronic active disease, and previous colonic resections were independently associated with the risk of developing MBD. Conclusions: The prevalence of MBD was significantly higher in CD patients. Besides the usual risk factors, we observed that factors related to chronic active and long-lasting disease increased the risk of MBD.

Dissecting neuronal development deficits by inflammation: from morphology to cytoskeleton dynamics

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Evaluation of Bone Remodeling in Hemodialysis Patients: Serum Biochemistry, Circulating Cytokines and Bone Histomorphometry

BACKGROUND: To optimize the noninvasive evaluation of bone remodeling, we evaluated, besides routine serum markers, serum levels of several cytokines involved in bone turnover. METHODS: A transiliac bone biopsy was performed in 47 hemodialysis patients. Serum levels of intact parathyroid hormone (iPTH; 1-84), total alkaline phosphatases (tAP), calcium, phosphate and aluminum (Al) were measured. Circulating levels of interleukin-6 (IL-6), IL-1 receptor antagonist (IL-1Ra) and soluble IL-6 receptor (sIL-6r) were determined using ELISA. Circulating IL-1beta, IL-6, IL-8, IL-10, IL-12p70 and tumor necrosis factor-alpha (TNF-alpha) were simultaneously quantified by flow cytometric immunoassay. RESULTS: Patients with low/normal bone formation rate (L/N-BFR) had significantly lower serum iPTH (p<0.001) and tAP (p<0.008) and significantly higher Al (p<0.025) than patients with high BFR. Serum calcium and phosphorus, however, did not differ (p=NS). An iPTH >300 pg/mL in association with tAP >120 U/L showed low sensitivity (58.8%) and low negative predictive value (44.0%) for the diagnosis of high BFR disease. An iPTH <300 pg/mL in association with normal or low tAP, <120 U/L, was associated with low sensitivity (66.7%) but high specificity (97.1%) for the diagnosis of L/N-BFR. Serum IL-1, IL-6, IL-12p70 and TNF-alpha were positively correlated with BFR, serum IL1-Ra and IL-10 with bone area, and by multiple regression analysis, tAP and IL-6 were independently predictive of BFR. CONCLUSIONS: Significant associations were found between several circulating cytokines and bone histomorphometry in dialysis patients. The usefulness of these determinations in the noninvasive evaluation of bone remodeling needs to be confirmed in larger dialysis populations.

Cytokine and nitric oxide production by mouse macrophages infected with brazilian flaviviruses

The Flaviviridae family, Flavivirus genus includes viruses that are transmitted to vertebrates by infected mosquitoes or ticks. The genus Flavivirus includes a variety of viruses that cause diseases such as acute febrile illness, encephalitis, and hemorrhagic fever. Flaviviruses primarily infect blood monocytes and tissue macrophages, which have been shown to be permissive, supporting viral replication and serving as virus reservoirs. On the other hand, these cells may have an important antiviral activity related to modulation by cytokine production and by the capacity of these cells to synthesize reactive free radicals such as nitric oxide (NO) which can have a microbicidal effect. The present study was performed in order to determine the production of cytokines interleukin-1beta (IL-1β), tumor necrosis factor -alpha (TNF-α), transforming growth factor- beta (TGF-β) and interferon -alpha (IFN-α) and NO by macrophages infected with one of four Brazilian flaviviruses, Bussuquara virus (BUSV), Yellow Fever virus (YFV), Rocio virus (ROCV) and Encephalitis Saint Louis virus (SLEV), and to verify the possible antiviral effect of NO during macrophage infection with ROCV. Moreover, we asked if the different viruses were able to regulate bacterial lipopolysaccharide (LPS) induced cytokine production. Our results showed that YFV and SLEV reduced the production of IL-1β and TGF-β by LPS-stimulated macrophages, while ROCV only diminished LPS-stimulated TGF-β synthesis. On the other hand, BUSV more likely favored an enhancement of the LPS-induced production of IL-1β by macrophages. Additionally, while most of the viruses stimulated the production of IFN-α, none of them altered the production of TNF-α by murine macrophages. Interestingly, all viruses induced synthesis of NO that was not correlated with antiviral activity for ROCV.

Homogneous time resolved fluorescence: a new technique for detection and quantifications of infliximab in autoimmune patients

RESUMO:As terapias biológicas revolucionaram o tratamento das doenças autoimunes nos últimos anos. Tipicamente têm como alvos mediadores importantes no mecanismo das doenças. Os antagonistas do fator de necrose tumoral-α (TNF-α) são um grupo de agentes biológicos muito prescrito, pois estão indicados no tratamento de doenças imuno-mediadas comuns, tais como artrite reumatoide, artrite idiopática juvenil, artrite psoriática, espondilite anquilosante, doença de Crohn e colite ulcerosa. Com o uso frequente de inibidores do TNF-α, tem-se tornado evidente que estes agentes têm um potencial imunogénico importante, que pode comprometer o prognóstico a longo prazo dos doentes cronicamente tratados. A produção de anticorpos anti-fármaco parece causar falência terapêutica secundária em muitos doentes. Um dos efeitos dos anticorpos anti-fármaco é o aumento da eliminação do fármaco. A eliminação do fármaco, por sua vez, varia entre indivíduos, refletindo diferentes perfis farmacocinéticos. A determinação dos níveis séricos mínimos do agente anti-TNF-α é assim muito informativa e pode auxiliar nas decisões terapêuticas. Contudo, os testes imunológicos para determinar as concentrações séricas do fármaco não estão facilmente disponíveis na prática clínica. De forma a investigar uma nova técnica potencialmente fidedigna e prática para a deteção e quantificação dos agentes biológicos anti-TNF-α, foi testada a técnica por HTRF (homogeneous time-resolved fluorescence resonance energy transfer) para a determinação de concentrações séricas de infliximab. Apesar de apresentar algumas limitações relacionadas com as condições de leitura da fluorescência, esta técnica provou obter resultados próximos das concentrações obtidas por ELISA (enzyme-linked immunosorbent assay) bridging. Adicionalmente, tem a vantagem de ser de execução muito mais fácil e rápida. Deste modo, a técnica por HTRF poderá ser otimizada e tornar-se uma valiosa ferramenta laboratorial para orientar as decisões terapêuticas em doentes autoimunes com falência da terapêutica anti-TNF-α.--------- ABSTRACT: Biologic therapies revolutionized the treatment of autoimmune diseases in the last years. Typically, they target important disease mediators. Tumor necrosis factor-alpha (TNF-α) antagonists constitute a very prescribed group of biologic agents as they are indicated for the treatment of common immune-mediated diseases, such as rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn’s disease and ulcerative colitis. With the increasing use of TNF-α inhibitors it has been noticed that they have an important immunogenic potential that can compromise long-term outcomes in chronically treated patients. The production of anti-drug antibodies seems to cause secondary therapeutic failure in many patients. One of the effects of anti-drug antibodies is the enhancement of drug clearance. Drug clearance, in turn, varies among individuals, reflecting different pharmacokinetic profiles. Determination of serum anti-TNF-α drug trough levels is though very informative and could support treatment decisions. However, immunologic assays to determine drug serum concentrations are not readily available in clinical practice. In order to investigate a potentially reliable and practical new technique for detection and quantification of anti-TNF-α biologic agents, homogeneous time-resolved fluorescence resonance energy transfer (HTRF) technique was tested for determination of serum infliximab concentrations. Although presenting some limitations related with fluorescence reading conditions, this technique proved to give results close to the concentrations obtained by the widely used bridging enzyme-linked immunosorbent assay (ELISA). In addition, it has the advantage of being much easier and faster to perform. Thus, HTRF technique can be optimized and become a valuable laboratorial tool to guide treatment decisions in autoimmune patients with anti-TNF-α therapy failure.

Clinical Outcomes and Genetic Expression Profile in Human Liver Graft Dysfunction During Ischemia/Reperfusion Injury

Introduction. This study aims to compare the molecular gene expression during ischemia reperfusion injury. Several surgical times were considered: in the beginning of the harvesting (T0), at the end of the cold ischemia period (T1), and after reperfusion (T2) and compared with graft dysfunction after liver transplant (OLT). Methods. We studied 54 patients undergoing OLT. Clinical, laboratory data, and histologic data (Suzuki classification) as well as the Survival Outcomes Following Liver Transplantation (SOFT) score were used and compared with the molecular gene expression of the following genes: Interleukin (IL)-1b, IL-6, tumor necrosis factor-a, perforin, E-selectin (SELE), Fas-ligand, granzyme B, heme oxygenase-1, and nitric oxide synthetase. Results. Fifteen patients presented with graft dysfunction according to SOFT criteria. No relevant data were obtained by comparing the variables graft dysfunction and histologic variables. We observed a statistically significant relation between SELE at T0 (P ¼ .013) and IL-1b at T0 (P ¼ .028) and early graft dysfunction. Conclusions. We conclude that several genetically determined proinflammatory expressions may play a critical role in the development of graft dysfunction after OLT.

Assessment by doppler ultrasound of entheseal lesions in spondyloarthritis : a longitudial study to determine structural damage and disease activity lesions

RESUMO: Enthesitis is the hallmark of spondyloarthritis (SpA), and is observed in all subtypes. Wide information on SpA abnormalities, including synovitis, tendinitis and enthesitis, can be efficiently perceived by Doppler ultrasound. Furthermore, several studies on imaging of enthesis showed that imaging techniques are better than clinical examination to detect enthesis alterations; and vascularized enthesitis detected by Doppler ultrasound appears to be a valuable diagnostic tool to confirm SpA diagnosis. However, data published until now concerning entheseal elementary alterations that characterize SpA enthesitis (enthesis inflammatory activity) or enthesopathy (permanent structural changes) reflect rather the authors’ empiric opinion than a methodological validation process. In this sense it seems crucial to identify elementary entheseal lesions associated with activity or damage, in order to improve monitoring and treatment response in SpA patients. The development of better assessment tools is today a challenge and a need in SpA. The first study of this thesis focused on the analysis of the reliability of inter-lector and inter-ultrasonography equipment of Madrid sonography enthesitis index (MASEI). Fundamental data for the remaining unrolling project validity. In the second and third studies we concerned about two entheseal elemental lesions: erosions and bursa. In literature erosions represent a permanent structural damage, being useful for monitoring joint injury, disease activity and therapeutic response in many rheumatic diseases; and to date, this concept has been mostly applied in rheumatoid arthritis (RA). Unquestionably, erosion is a tissue-related damage and a structural change. However, the hypothesis that we decided to test was if erosions represent a permanent structural change that can only grow and worsen over time, as occurs in RA, or a transitory alteration. A longitudinal study of early SpA patients was undertaken, and the Achilles enthesis was used as a model. Our results strongly suggested that previously detected erosions could disappear during the course of the disease, being consistent with the dynamic behavior of erosion over time. Based on these striking results it seems reasonable to suggest that the new-bone formation process in SpA could be associated with the resolution of cortical entheseal erosion over time. These results could also be in agreement with the apparent failure of anti-tumor necrosis factor (TNF) therapies to control bone proliferation in SpA; and with the relation of TNF-α, Dickkopf-related protein 1 (Dkk-1) and the regulatory molecule of the Wnt signaling pathway in the bone proliferation in SpA. In the same model, we then proceeded to study the enthesis bursa. Interestingly, the Outcome Measures in Rheumatology Clinical Trials (OMERACT) enthesopathy definition does not include bursa as an elementary entheseal lesion. Nonetheless, bursa was included in 46% of the enthesis studies in a recently systematic literature review, being in agreement with the concept of “synovio-entheseal complex” that includes the link between enthesitis and osteitis in SpA. It has been clarified in recent data that there is not only a close functional integration of the enthesis with the neighboring bone, but also a connection between enthesitis and synovitis. Therefore, we tried to assess the prevalence and relevance of the bursa-synovial lesion in SpA. Our findings showed a significant increase of Achilles bursa presence and thickness in SpA patients compared to controls (healthy/mechanical controls and RA controls). These results raise awareness to the need to improve the enthesopathy ultrasonographic definition. In the final work of this thesis, we have explored new perspectives, not previously reported, about construct validity of enthesis ultrasound as a possible activity outcome in SpA. We performed a longitudinal Achilles enthesis ultrasound study in patients with early SpA. Achilles ultrasound examinations were performed at baseline, six- and twelve-month time periods and compared with clinical outcome measures collected at basal visit. Our results showed that basal erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are higher in patients with Doppler signal in enthesis, and even that higher basal ESR, CRP and Ankylosing Spondylitis Disease Activity Score (ASDAS) predicted a higher Doppler signal (an ultrasound alteration accepted as representative of inflammation) six months later. Patients with very high disease activity assessed by ASDAS (>3.5) at baseline had significantly higher Achilles total ultrasound score verified at the same time; and ASDAS <1.3 predicted no Doppler signal at six and twelve months. This seems to represent a connection between classical biomarkers and clinical outcomes associated with SpA activity and Doppler signal, not only at the same time, but also for the following months. Remarkably, patients with inactive disease (ASDAS < 1.3) at baseline had no Doppler signal at six and twelve months. These findings reinforce the potential use of ultrasound related techniques for disease progression assessment and prognosis purposes. Intriguingly, Ankylosing Spondylitis Disease Activity Index (BASDAI) didn’t show significant differences between different cut-offs concerning ultrasound lesions or Doppler signal, while verified with ASDAS. These results seem to indicate that ASDAS reflects better than BASDAI what happens in the enthesis. The work herein discussed clearly shows the potential utility of ultrasound in enthesis assessment in SpA patients, and can be important for the development of ultrasound activity and structural damage scores for diagnosis and monitoring purposes. Therefore, local promotion of this technique constitutes a medical intervention that is worth being tested in SpA patients for diagnosis, monitoring and prognosis purposes.

Plasma levels of immunoinflammatory markers in De Novo coronary atherosclerosis and coronary restenosis postangioplasty

OBJECTIVE: To compare circulating plasma levels of immunoinflammatory markers in patients with known de novo coronary artery disease and patients with postangioplasty restenosis. METHODS: Using enzymatic immunoabsorbent assay, we measured plasma levels of soluble interleukin-2 receptosr, tumor necrosis factor alpha, and soluble tumor necrosis alpha receptors I and II in 11 patients with restenosis postcoronary angioplasty (restenosis group), in 10 patients with primary atherosclerosis (de novo group) who were referred for coronary angiography because of stable or unstable angina, and in 9 healthy volunteers (control group). Levels of soluble interleukin-2 receptors were significantly higher in the de novo group compared with that in the restenosis and control groups. Levels were also higher in the restenosis group compared with that in the control group. Plasma levels of tumor necrosis alpha and receptor levels were significantly higher in the de novo group compared to with that in the restenosis and control groups, but levels in the restenosis group were not different from that in the controls. CONCLUSION: Coronary artery disease, either primary or secondary to restenosis, is associated with significant immunoinflammatory activity, which can be assessed by examining the extent of circulating plasma levels of inflammatory markers. Moreover, patients with de novo lesions appear to have increased inflammatory activity compared with patients with restenosis.