Relevance of the deletion polymorphisms of the glutathione S-transferases GSTT1 and GSTM1 in pharmacology and toxicology

Although cytosolic glutathione S-transterase (GST) enzymes occupy a key position in biological detoxification processes, two of the most relevant human isoenzymes. GST1-1 and GSTM1-1, are genetically deleted (non-functional alleles GSTT1*0 and GsTM1*0) in a high percentage of the human population, with major ethnic differences. The structures of the GSTT and GSTM gene areas explain the underlying genetic processes. GSTT1-1 is highly conserved during evolution and plays a major role in phase-II biotransformation of a number of drugs and industrial chemicals. e.g. cytostatic drugs, hydrocarbons and halogenated hydrocarbons. GSTM1-1 is particularly relevant in the deactivation of carcinogenic intermediates of polycyclic aromatic hydrocarbons. Several lines of evidence Suggest that hGSTT1-1 and/or hGSTM1-1 play a role in the deactivation of reactive oxygen species that are likely to be involved in cellular processes of inflammation, ageing and degenerative diseases. There is cumulating evidence that combinations of the GSTM1*0 state with other genetic traits affecting the metabolism of carcinogens (CYP1A1, GSTP1) may predispose the aero-digestivc tract and lung, especially in smokers, to a higher risk of cancer. The GSTM1*0 status appears also associated with a modest increase in the risk of bladder cancer, consistent with a GSTM1 interaction with carcinogenic tobacco smoke constituents. Both human GST deletions, although largely counterbalanced by overlapping substrate affinities within the GST superfamily, have consequences when the organism comes into contact with distinct man-made chemicals. This appears relevant in industrial toxicology and in drug metabolism.

DNA Motifs Suppressing TLR9 Responses

Immune cells respond to bacterial DNA containing unmethylated CpG motifs via Toll-like receptor 9 (TLR9). Given the apparent role of TLR9 in development of systemic lupus erythernatosus (SLE), there is interest in the development of TLR9 inhibitors. TLR9-mediated responses are reported to be inhibited by a confusing variety of different DNA sequences and structures. To aid characterization, we have provisionally categorized TLR9-inhibitory oligodeoxynucleoti des (ODN) into 4 classes, on the basis of sequence and probable mode of action. Class I are short G-rich ODN, which show sequence-specific inhibition of all TLR9 responses, and may be direct competitive inhibitors for DNA binding to TLR9. Class II are telomeric repeat motifs that inhibit STAT signaling, and thus are not specific to TLR9 responses. Because Class II ODN are generally made as 24-base phosphorothioate-modified ODN (PS-ODN), they also fall into Class IV, defined as long PS-ODN, which inhibit TLR9 responses in a sequence-nonspecific manner. Class III includes oligo (dG) that forms a 4-stranded structure and inhibits DNA uptake. The Class I G-rich motifs show the most promise as selective and potent TLR9 inhibitors for therapeutic applications.

Is the generation of neo-antigenic determinants by free radicals central to the development of autoimmune rheumatoid disease?

Biomolecules are susceptible to many different post-translational modifications that have important effects on their function and stability, including glycosylation, glycation, phosphorylation and oxidation chemistries. Specific conversion of aspartic acid to its isoaspartyl derivative or arginine to citrulline leads to autoantibody production in models of rheumatoid disease, and ensuing autoantibodies cross-react with native antigens. Autoimmune conditions associate with increased activation of immune effector cells and production of free radical species via NADPH oxidases and nitric oxide synthases. Generation of neo-antigenic determinants by reactive oxygen and nitrogen species ROS and RNS) may contribute to epitope spreading in autoimmunity. The oxidation of amino acids by peroxynitrite, hypochlorous acid and other reactive oxygen species (ROS) increases the antigenicity of DNA, LDL and IgG, generating ligands for which autoantibodies show higher avidity. This review focuses on the evidence for ROS and RNS in promoting the autoimmune responses observed in diseases rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). It considers the evidence for ROS/RNS-induced antigenicity arising as a consequence of failure to remove or repair ROS/RNS damaged biomolecules and suggests that an associated defect, probably in T cell signal processing or/or antigen presentation, is required for the development of disease.

Probing molecular changes induced in DNA by reactive oxygen species with monoclonal antibodies

Antibodies reactive with native double stranded DNA are characteristic of the chronic inflammatory disease systemic lupus erythematosus. Native DNA is however, a poor immunogen and the mechanism of anti-DNA antibody production is incompletely understood. Modification of DNA can increase its immunogenicity and in inflammatory disease states reactive oxygen species produced from phagocytic cells have been shown to thus modify DNA. In this study, monoclonal antibodies produced spontaneously by two mice strains with lupus-like disease were used in a competition ELISA to monitor changes to DNA induced by reactive oxygen species. Different procedures for reactive oxygen species generation were found to cause distinct and characteristic changes to DNA involving modifications of base residues, the sugar-phosphate backbone and the gross conformational structure of double-stranded DNA. In view of this, it may be possible to use these antibodies further to probe DNA and infer the source and nature of the reactive oxygen species it has been exposed to, particularly in vivo.

Reactive oxygen species induce antigenic changes in DNA

Reactive oxygen species (ROS) are released at sites of inflammation during the respiratory burst which accompanies the phagocytic process. Using an in vitro system to simulate this process we have shown that ROS induce antigenic changes in DNA. More specifically, results of experiments using ROS scavengers have shown that hydroxyl radicals produced in close proximity to DNA-bound metal ions play a predominant role. ROS-mediated attack resulted in increased binding of anti-DNA antibodies to the denatured DNA. These changes were detected using IgG, IgA and IgM isotype binding to antibodies in systemic lupus erythematosus sera. Of these the IgA isotype was most discriminating in its detection of hydroxyl radical-induced damage.

Innate immunity and apoptosis:CD14-dependent clearance of apoptotic cells

This is the first comprehensive book about the relationship between apoptosis and autoimmune diseases. It offers a unique up–to–date overview on research results on the defective execution of apoptosis and the incomplete clearance of apoptotic cells. The molecular and cellular mechanisms involved are described in detail. As a possible consequence of apoptotic dysfunction, the development of severe autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus) is discussed. An outlook on future research topics includes the evaluation of novel therapeutic strategies.

Mulheres com lúpus eritematoso sistêmico: sintomas depressivos e apoio social percebido

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease, rare, multisystem, with a very heterogeneous clinical and serological manifestations standard. The patient, in addition to suffering injuries on his physical and physiological functioning, may also face a number of psychosocial problems. Research indicates that SLE can cause significant damage to the psychological realm, especially with the presence of anxiety and depression. In 1999, the American College of Rheumatology (ACR), proposed the establishment of 19 neuropsychiatric clinical syndromes attributed to SLE. Depression lies between mood disorders and is one of the most common psychiatric manifestations in this group, being found more frequently in these patients than in the general population. Studies also suggest that social support plays an important role in the development of coping strategies, in SLE management and depression. This study has as main objective verify the association between depressive symptoms and perceived social support in patients with SLE. The specific objectives turned to: investigte the prevalence of depressive symptoms; investigate the perceived social support and verify if there is an association between depression, social support and sociodemographic variables. We used a sociodemographic questionnaire, the Beck Depression Scale, and the Perceived Social Support Scale. The analysis was performed through descriptive and inferential statistics. The final sample could count with 79 SLE women, with an average age of 35.7 years. 44 (55.7%) of the participants were married. Only 6 (7.59%) had completed higher education and 32 (40.51%) have not finished high school. Seventy-one (89.87%) had an income below three minimum salaries and 71 (89.87) practiced a religion, and the Catholic (67.71%) was the most mentioned by them. Of the total sample, 37 (46.74%) had been diagnosed SLE more than 7 years before, and 25 (31.65%) had the disease for more than 10 years. Only 19 (24.05%) had some work activity. Forty-two of them (53.17%) had depressive symptoms levels from mild to severe, and 51 (64.46%) reported pain levels of 5, or above. The study found a significant association between depressive symptoms and pain (p = 0.013) and depressive symptoms and work activity (p = 0.02). When we examined the perception of social support, the results showed high levels among participants. Using the Spearman correlation test we found a strong correlation between depressive symptoms and social support (p= 0,000037). It means that the higher the frequency of support, the lower the score of depression. These findings are relevant because depressive symptoms in patients with SLE have a multicausal and multifactorial character and may remain unnoticed, since many of them are confused with the manifestations of the disease. This fact requires a careful assessment from professionals, not only in the clinical setting, but also considering other psychosocial reasons, that may be influencing the emergence or worsening of symptoms. These results also corroborate other studies, which not only confirm the predictive role of social support in the physical wellbeing, but also in the psychological.

Mulheres com lúpus eritematoso sistêmico: sintomas depressivos e apoio social percebido

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease, rare, multisystem, with a very heterogeneous clinical and serological manifestations standard. The patient, in addition to suffering injuries on his physical and physiological functioning, may also face a number of psychosocial problems. Research indicates that SLE can cause significant damage to the psychological realm, especially with the presence of anxiety and depression. In 1999, the American College of Rheumatology (ACR), proposed the establishment of 19 neuropsychiatric clinical syndromes attributed to SLE. Depression lies between mood disorders and is one of the most common psychiatric manifestations in this group, being found more frequently in these patients than in the general population. Studies also suggest that social support plays an important role in the development of coping strategies, in SLE management and depression. This study has as main objective verify the association between depressive symptoms and perceived social support in patients with SLE. The specific objectives turned to: investigte the prevalence of depressive symptoms; investigate the perceived social support and verify if there is an association between depression, social support and sociodemographic variables. We used a sociodemographic questionnaire, the Beck Depression Scale, and the Perceived Social Support Scale. The analysis was performed through descriptive and inferential statistics. The final sample could count with 79 SLE women, with an average age of 35.7 years. 44 (55.7%) of the participants were married. Only 6 (7.59%) had completed higher education and 32 (40.51%) have not finished high school. Seventy-one (89.87%) had an income below three minimum salaries and 71 (89.87) practiced a religion, and the Catholic (67.71%) was the most mentioned by them. Of the total sample, 37 (46.74%) had been diagnosed SLE more than 7 years before, and 25 (31.65%) had the disease for more than 10 years. Only 19 (24.05%) had some work activity. Forty-two of them (53.17%) had depressive symptoms levels from mild to severe, and 51 (64.46%) reported pain levels of 5, or above. The study found a significant association between depressive symptoms and pain (p = 0.013) and depressive symptoms and work activity (p = 0.02). When we examined the perception of social support, the results showed high levels among participants. Using the Spearman correlation test we found a strong correlation between depressive symptoms and social support (p= 0,000037). It means that the higher the frequency of support, the lower the score of depression. These findings are relevant because depressive symptoms in patients with SLE have a multicausal and multifactorial character and may remain unnoticed, since many of them are confused with the manifestations of the disease. This fact requires a careful assessment from professionals, not only in the clinical setting, but also considering other psychosocial reasons, that may be influencing the emergence or worsening of symptoms. These results also corroborate other studies, which not only confirm the predictive role of social support in the physical wellbeing, but also in the psychological.

Delivery and Scavenging of Nucleic Acids by Polycationic Polymers

Electrostatic interaction is a strong force that attracts positively and negatively charged molecules to each other. Such an interaction is formed between positively charged polycationic polymers and negatively charged nucleic acids. In this dissertation, the electrostatic attraction between polycationic polymers and nucleic acids is exploited for applications in oral gene delivery and nucleic acid scavenging. An enhanced nanoparticle for oral gene delivery of a human Factor IX (hFIX) plasmid is developed using the polycationic polysaccharide, chitosan (Ch), in combination with protamine sulfate (PS) to treat hemophilia B. For nucleic acid scavenging purposes, the development of an effective nucleic acid scavenging nanofiber platform is described for dampening hyper-inflammation and reducing the formation of biofilms.

Non-viral gene therapy may be an attractive alternative to chronic protein replacement therapy. Orally administered non-viral gene vectors have been investigated for more than one decade with little progress made beyond the initial studies. Oral administration has many benefits over intravenous injection including patient compliance and overall cost; however, effective oral gene delivery systems remain elusive. To date, only chitosan carriers have demonstrated successful oral gene delivery due to chitosan’s stability via the oral route. In this study, we increase the transfection efficiency of the chitosan gene carrier by adding protamine sulfate to the nanoparticle formulation. The addition of protamine sulfate to the chitosan nanoparticles results in up to 42x higher in vitro transfection efficiency than chitosan nanoparticles without protamine sulfate. Therapeutic levels of hFIX protein are detected after oral delivery of Ch/PS/phFIX nanoparticles in 5/12 mice in vivo, ranging from 3 -132 ng/mL, as compared to levels below 4 ng/mL in 1/12 mice given Ch/phFIX nanoparticles. These results indicate the protamine sulfate enhances the transfection efficiency of chitosan and should be considered as an effective ternary component for applications in oral gene delivery.

Dying cells release nucleic acids (NA) and NA-complexes that activate the inflammatory pathways of immune cells. Sustained activation of these pathways contributes to chronic inflammation related to autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, and inflammatory bowel disease. Studies have shown that certain soluble, cationic polymers can scavenge extracellular nucleic acids and inhibit RNA-and DNA-mediated activation of Toll-like receptors (TLRs) and inflammation. In this study, the cationic polymers are incorporated onto insoluble nanofibers, enabling local scavenging of negatively charged pro-inflammatory species such as damage-associated molecular pattern (DAMP) molecules in the extracellular space, reducing cytotoxicity related to unwanted internalization of soluble cationic polymers. In vitro data show that electrospun nanofibers grafted with cationic polymers, termed nucleic acid scavenging nanofibers (NASFs), can scavenge nucleic acid-based agonists of TLR 3 and TLR 9 directly from serum and prevent the production of NF-ĸB, an immune system activating transcription factor while also demonstrating low cytotoxicity. NASFs formed from poly (styrene-alt-maleic anhydride) conjugated with 1.8 kDa branched polyethylenimine (bPEI) resulted in randomly aligned fibers with diameters of 486±9 nm. NASFs effectively eliminate the immune stimulating response of NA based agonists CpG (TLR 9) and poly (I:C) (TLR 3) while not affecting the activation caused by the non-nucleic acid TLR agonist pam3CSK4. Results in a more biologically relevant context of doxorubicin-induced cell death in RAW cells demonstrates that NASFs block ~25-40% of NF-ĸβ response in Ramos-Blue cells treated with RAW extracellular debris, ie DAMPs, following doxorubicin treatment. Together, these data demonstrate that the formation of cationic NASFs by a simple, replicable, modular technique is effective and that such NASFs are capable of modulating localized inflammatory responses.

An understandable way to clinically apply the NASF is as a wound bandage. Chronic wounds are a serious clinical problem that is attributed to an extended period of inflammation as well as the presence of biofilms. An NASF bandage can potentially have two benefits in the treatment of chronic wounds by reducing the inflammation and preventing biofilm formation. NASF can prevent biofilm formation by reducing the NA present in the wound bed, therefore removing large components of what the bacteria use to develop their biofilm matrix, the extracellular polymeric substance, without which the biofilm cannot develop. The NASF described above is used to show the effect of the nucleic acid scavenging technology on in vitro and in vivo biofilm formation of P. aeruginosa, S. aureus, and S. epidermidis biofilms. The in vitro studies demonstrated that the NASFs were able to significantly reduce the biofilm formation in all three bacterial strains. In vivo studies of the NASF on mouse wounds infected with biofilm show that the NASF retain their functionality and are able to scavenge DNA, RNA, and protein from the wound bed. The NASF remove DNA that are maintaining the inflammatory state of the open wound and contributing to the extracellular polymeric substance (EPS), such as mtDNA, and also removing proteins that are required for bacteria/biofilm formation and maintenance such as chaperonin, ribosomal proteins, succinyl CoA-ligase, and polymerases. However, the NASF are not successful at decreasing the wound healing time because their repeated application and removal disrupts the wound bed and removes proteins required for wound healing such as fibronectin, vibronectin, keratin, and plasminogen. Further optimization of NASF treatment duration and potential combination treatments should be tested to reduce the unwanted side effects of increased wound healing time.

Health-related quality of life in portuguese SLE patients: an outcome measure independent of disease activity and cumulative damage

PURPOSE: To evaluate quality of life in Portuguese patients with Systemic Lupus Erithematosus (SLE) and its correlation with disease activity and cumulative damage. METHODS: We included consecutive SLE patients, fulfilling the 1997 ACR Classification Criteria for SLE and followed at the Rheumatology Department of the University Hospital of Coimbra, Portugal at time of visit to the outpatient clinic. Quality of life was evaluated using the patient self-assessment questionnaire Medical Outcomes Survey Short Form-36 (SF-36) (validated Portuguese version). The consulting rheumatologist fulfilled the SLE associated indexes for cumulative damage (Systemic Lupus International Collaborating Clinics- Damage Index: SLICC/ACR-DI) and disease activity (Systemic Lupus Erythematosus Disease Activity Index: SLEDAI 2000). Correlation between SLEDAI and SLICC and SF-36 was tested with the Spearman Coefficient. Significant level considered was 0.05. RESULTS: The study included 133 SLE patients (90.2% female, mean age - 40.7 years, mean disease duration - 8.7 years). Most patients presented low disease activity (mean SLEDAI = 4.23) and limited cumulative damage (mean SLICC = 0.76). Despite that, SF-36 mean scores were below 70% in all eight domains of the index. Physical function domains showed lower scores than mental function domains. The QoL in this group of patients is significantly impaired when compared with the reference Portuguese population (p<0.05 in all domains). There was no correlation between clinical activity or cumulative damage and quality of life. CONCLUSION: QoL is significantly compromised in this group of SLE patients, but not related with disease activity or damage. These findings suggest that disease activity, cumulative damage and QoL are independent outcome measures and should all be used to assess the full impact of disease in SLE patients.

Auricular Chondritis in a Postpartum Flare of SLE and APS

Introduction: Auricular chondritis has been occasionally described in patients with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). Materials and methods: We report the case of a woman with a previous history of APS who presented with auricular chondritis with onset of SLE symptoms during the postpartum period. Conclusion: SLE and APS should be taken into consideration in the differential diagnosis of auricular chondritis.

Construção e produção de fragmentos de anticorpos anti-ssDNA em um contexto de desenvolvimento de linfócitos B

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2016.

Factores asociados a la mortalidad en el síndrome de Sjögren

Antecedentes: El síndrome de Sjögren (SS) es una patología crónica, autoinmune, de características multifactoriales en su etiología. También es conocida como una epitelitis autoinmune, caracterizada por síntomas secos como xeroftalmia y xerostomía, pero que también puede tener compromiso sistémico, dado por manifestaciones extra-glandulares. En la actualidad es poco reconocida como tal, y por lo tanto, la tasa de sobrevida en estos pacientes se encuentra disminuida pero poco tenida en cuenta a la hora de la valoración de ellos. Este trabajo describe la evidencia encontrada acerca de las causas de mortalidad y sus factores asociados luego de realizar una revisión sistemática de la literatura. Objetivos: El objetivo de este estudio fue reunir de forma exhaustiva y sistemática toda la evidencia empírica, publicada o no, que cumpla los criterios de búsqueda y elegibilidad sobre factores asociados al incremento de la mortalidad o disminución en la sobrevida de los pacientes con diagnóstico de SS. Métodos: Se realizó una revisión sistemática de la literatura mediante una búsqueda exhaustiva de todos los estudios publicados en las bases de datos electrónicas preestablecidas, hasta abril de 2015, con el fin de determinar las causas más frecuentes de mortalidad en pacientes con SS y los factores asociados a ella. Resultados: Se encontraron 4,654 resultados que coincidían con los criterios de búsqueda establecidos; de estos, 33 cumplieron con los criterios de inclusión y se distribuyeron de la siguiente forma: el 66.6% (22/33) correspondieron a estudios de corte cohorte, 30.3% (10/33) a estudios de corte transversal y el 3.03% (1/33) a estudios casos y controles. Se obtuvieron resultados en cuanto a frecuencias de mortalidad, razón estandarizada de mortalidad, tasas de supervivencia, causas más frecuentes de mortalidad y sus factores asociados. Conclusiones: La mortalidad reportada en los diferentes estudios fue entre el 1.2% hasta el 30%. Aquellos estudios que reportaron una tasa de mortalidad inferior al 5%, tuvieron un tiempo de seguimiento menor 8 años [1,7,33,60,64,86]. La mayoría de los casos sigue un curso relativamente estable, pero hay un porcentaje importante que presenta otras manifestaciones sistémicas con mayor frecuencia de complicaciones durante la evolución del SS. Por tanto, son los que requieren un seguimiento más estrecho, debido a una mayor necesidad de tratamiento sistémico y al mayor riesgo de ingreso hospitalario y de mortalidad, especialmente por el desarrollo de procesos linfoproliferativos B. La presencia de factores pronósticos en el paciente con SS obligará a realizar un seguimiento clínico e inmunológico mucho más estrecho, lo cual permitirá identificar lo antes posible las complicaciones que puedan aparecer e instaurar las correspondientes medidas terapéuticas, para aumentar las tasas de supervivencia.